Methods of using monoclonal antibodies targeting epitopes of asph

ABSTRACT

Monoclonal antibodies (MAbs) targeting one or more specific epitopes of aspartyl (asparaginyl) hydroxylase (ASPH), including humanized, bi-specific, and other chimeric MAb variants, and fragments thereof (collectively ASPH epitope-specific MAbs, or simply ASPH MAbs), are disclosed. Methods of production, purification, and use of the ASPH epitope-specific MAbs, and compositions comprising them, as agents in therapeutic and diagnostic applications to interact with target molecules in cell-free samples, cell- and tissue-based assays, animal models, and in a subject are also disclosed. Other aspects of the invention relate to use of the molecules disclosed herein to diagnose, ameliorate, or treat cell proliferation disorders and related diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application that claims priority to and thebenefit of U.S. Non-Provisional application Ser. No. 17/036,337, filedSep. 29, 2020, issued as U.S. Pat. No. 11,236,178 on Feb. 1, 2022, U.S.Non-Provisional application Ser. No. 16/444,617, filed Jun. 18, 2019,issued as U.S. Pat. No. 10,961,320 on Mar. 30, 2021 and U.S. ProvisionalApplication No. 62/686,107, filed Jun. 18, 2018, the entire contents ofwhich are incorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF A SEQUENCE LISTING

The Sequence Listing contained in the files“761_190_045_US_01_Sequence_Listing_ST25.txt”, created on 2022-01-25,modified on 2022-01-25, file size 44,400 bytes, containing SEQ ID NOS:1-52, “761_190_033_US_01_Sequence_Listing_ST25.txt”, created on2020-09-01, modified on 2020-09-01, file size 44,370 bytes, containingSEQ ID NOS: 1-52, “761_190_026_US_Sequence_Listing_ST25.txt”, created on2019-05-21, modified on 2019-05-21, file size 35,033 bytes, containingSEQ ID NOS: 1-30, and “761_190_025_US_Sequence_Listing_ST25.txt”,created on 2018-06-17, modified on 2018-06-17, file size 34,990 bytes,containing SEQ ID NOS: 1-30, are incorporated by reference in itsentirety herein.

FIELD OF THE INVENTION

Monoclonal antibodies (MAbs) targeting one or more specific epitopes ofaspartyl (asparaginyl) β-hydroxylase (ASPH), including humanized,bi-specific, and other chimeric MAb variants, and fragments thereof(collectively ASPH epitope-specific MAbs, or simply ASPH MAbs), aredisclosed. Methods of production, purification, and use of the ASPHepitope-specific MAbs, and compositions comprising them, as agents intherapeutic and diagnostic applications to interact with targetmolecules in cell-free samples, cell- and tissue-based assays, animalmodels, and in a subject are also disclosed. Other aspects of theinvention relate to use of the molecules disclosed herein to diagnose,ameliorate, or treat cell proliferation disorders and related diseases.

BACKGROUND OF THE INVENTION

Aspartyl(asparaginyl)-β-hydroxylase (ASPH) is an iron-dependentdioxygenase that catalyzes the hydroxylation of β carbons of asparticacid and asparagine residues in calcium binding Epidermal Growth Factor(cbEGF)-like domains of a variety of proteins, including Notch and Notchligand homologs (Dinchuk, Focht et al. 2002) extracellular matrixproteins, and low density lipoprotein (LDL) receptors. ASPH was firstobserved to be involved in the hydroxylation of a specific aspartic acidresidue in the blood coagulation cascade proteins (Drakenberg, Fernlundet al. 1983) where the hydroxylated residue is underlined in theconsensus sequence CX[D/N]X₄[Y/F]XC. The role of hydroxylated residue ispresently unknown, but the sole known crystal structure with abeta-hydroxylated asparagine (PDB ID 5JZZ: McDonough, M. A., Pfeffer,I., and Munzel (2016) Aspartyl/Asparaginyl beta-hydroxylase(AspH)oxygenase and TPR domains in complex with manganese,N-oxalylglycine and cyclic peptide substrate mimic of factor X. DOI:10.2210/pdb5JZZ/pdb).

ASPH is generally classified as a peptide-aspartate beta-dioxygenase (EC1.14.11.16), a member of the alpha-ketoglutarate-dependent hydroxylasessuperfamily, which catalyzes the following chemical reaction,facilitated by iron as a cofactor.

peptide-L-aspartate+2-oxoglutarate+O₂→peptide-3-hydroxy-L-aspartate+succinate+CO₂  (Reaction1)

ASPH is not normally expressed in adult cells (Lavaissiere, Jia et al.1996), but is expressed during invasion of the uterine wall bytrophoblasts during development of the placenta (Gundogan, Elwood et al.2007). ASPH is overexpressed in a variety of tumors, includinghepatocellular, cholangiocarcinoma, gastric cancer, pancreatic cancer,non-small cell lung cancer, glioblastoma multiform, osteosarcoma,cervical cancer, ovarian cancer and breast cancer (Yang, Song et al.2010), and enhances signaling in the Notch pathway (Cantarini, de laMonte et al. 2006).

FIG. 1 sets forth an illustration showing the Activation of NotchSignaling Pathway by ASPH. FIGS. 2 and 3 set forth illustrations showingthe Locations of Epitopes of Interest on ASPH.

Known and computationally predicted ASPH substrates are illustrated inFIG. 4 and FIG. 5. Prediction of ASPH substrates is based upon theprotein possessing A) a cbEGF domain and B) the consensus sequenceCX[D/N]X₄[Y/F]XC. Of particular interest are nearly all of the Notchsignaling proteins, not only including the receptors Notch1-4, but manyof the known ligands such as Jagged1&2 and DII1&4, but also known Notchpathway modulator human homologues of Crumbs from Drosophila. ASPH isknown to hydroxylate lipid receptor proteins, including Lrp1. Lrp1 isknown to have an interaction with Wnt5a of the canonical Wnt signalingpathway (El Asmar, Terrand et al. 2016). ASPH substrate Gas6 is theligand of the Tyro3, Axel and Mer (TAM) kinases, which have beenimplicated in cancer (Wu, Ma et al. 2018). Known ASPH substratesincluding the fibrillins are involved in the release of TGF-beta, whichis implicated in cancer (Furler, Nixon et al. 2018). In addition tocancer, ASPH hydroxylated substrates are found in nearly all of theblood coagulation proteins involved in thrombosis (see panel C in FIG.4), and many of the proteins involved in lipid uptake including LDLR,VLDLR and Lrp1 (see panel B in FIG. 4) and cholesterol homeostasis.Thus, ASPH expression is expected to have a cascade of effects, but mayhave particular value in the treatment of cancer, as well as thrombosisand lipid/cholesterol associated cardiovascular diseases.

ASPH is known to contain multiple phosphorylation sites (Tong, Gao etal. 2013), including T748. Phosphorylation of ASPH is known to alter theexpression and function of ASPH (Borgas, Gao et al. 2015), and plays apotential role in migration and tissue invasion of hepatocellularcarcinoma (Borgas, Gao et al. 2015). Antibodies selective for ASPHphosphorylation state should be useful in the diagnosis of cancer anddistinguishing normally expressed ASPH from tumor expressed ASPH.

Previously designed antibodies to ASPH did not result in directsuppression of tumor cell proliferation (Yeung, Finney et al. 2007).Despite the high affinity of these antibodies, the targeted epitope didnot sufficiently disrupt catalytic activity of ASPH. Consequently, whilethe antibodies were internalized into the cancer cells expressing ASPH,there was no direct antibody activity leading to cellular senescence orcytotoxicity. To address this issue, radioisotopes have been conjugatedto previously described high affinity ASPH antibodies, leading to modestactivity (Revskaya, Jiang et al. 2017). Other previous anti-ASPHstrategies include small molecule inhibitors of ASPH (Aihara, Huang etal. 2014), a dendritic cell approach (Noda, Shimoda et al. 2012), and avaccine approach (Iwagami, Casulli et al. 2017).

This application describes the epitope selection for phospho-selectiveASPH antibodies, as well as antibodies for ASPH catalytic activityinhibition, including epitopes on both the catalytic and non-catalyticdomains, demonstration of high affinity for ASPH, strong IHC staining ofcancerous but not normal tissue, and direct activity against cancercells.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies (MAbs) targetingone or more specific epitopes of aspartyl (asparaginyl) β-hydroxylase(ASPH), including chimeric and humanized MAb variants, and fragmentsthereof (collectively ASPH epitope-specific MAbs, or simply ASPH MAbs),are disclosed. Methods of production, purification, and use of the ASPHepitope-specific MAbs, and compositions comprising them, as agents intherapeutic and diagnostic applications to interact with targetmolecules in cell-free samples, cell- and tissue-based assays, animalmodels, and in a subject are also disclosed. Other aspects of theinvention relate to use of the molecules disclosed herein to diagnose,ameliorate, or treat cell proliferation disorders and related diseases.

One aspect relates to an isolated monoclonal antibody, or a fragmentthereof, which binds to a one or more peptide epitopes of human aspartyl(asparaginyl) β-hydroxylase (ASPH), wherein at least one of said peptideepitopes is located within or adjacent to the catalytic domain of ASPH.

Another aspect relates to a composition comprising any of the antibodiesnoted above, including compositions comprising at least one antibodythat targets ASPH and one or more pharmaceutical excipients.

Another aspect relates to a method of using any of the antibodies notedabove, to inhibit the proliferation of isolated tumor cell samples grownin culture.

Another aspect relates to a method of using any of the antibodies notedabove, to inhibit the proliferation of tumor cells in tissue samplesgrown in culture.

Another aspect relates to a method of treating cancer in a mammaliansubject, comprising administering to a subject in need thereof anantibody as noted above in an amount sufficient to treat cancer.

Another aspect relates to a kit for diagnosis of cancer in a mammaliansubject, wherein said kit comprises an antibody, or a fragment thereof,of any of any of the antibodies noted above.

Another aspect relates to a humanized antibody comprising one or morecomplementarity determining regions (CDRs) derived from a non-humansource targeting one or more peptide epitopes located within or adjacentto the catalytic domain of ASPH of any of the antibodies noted above,and one or more portions of the constant regions of a human antibody,and fragments thereof.

Another aspect relates to a bispecific antibody comprising one or morecomplementarity determining regions (CDRs) derived from a non-humansource targeting one or more peptide epitopes located within or adjacentto the catalytic domain of ASPH of any of the antibodies noted above,and an antibody targeting other epitopes selected from the groupconsisting of the T-cell redirector class, comprising an antibodytargeting one or more ASPH CDRs and an antibody targeting CD3; theNK-cell redirector class, comprising an antibody targeting one or moreASPH CDRs and an antibody targeting CD16A; the tumor targetingimmunomodular class, comprising an antibody targeting one or more ASPHCDRs and an antibody targeting CD40 or 4-1BB; and the dual immunomodularclass, comprising an antibody targeting one or more ASPH CDRs and anantibody targeting PD-L1, PD-1, CTLA-4, TGF-0, LAG-3, TIM-3, or OX40.

A better understanding of the invention will be obtained from thefollowing detailed descriptions and accompanying drawings, which setforth illustrative embodiments that are indicative of the various waysin which the principals of the invention may be employed.

BRIEF DESCRIPTION OF THE DRAWINGS Statement Concerning Aspects of theInvention Understood by Reference to the Drawings

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 sets forth an illustration showing the Activation of NotchSignaling Pathway by ASPH. Aspartyl(asparaginyl)-β-hydroxylase (ASPH) isan iron-dependent dioxygenase that catalyzes the hydroxylation of βcarbons of aspartic acid and asparagine residues in domains of a varietyof proteins, including Notch and Notch ligand homologs. Intenseactivation of the Notch pathway by ASPH is observed in tumor tissues.Inhibition of ASPH allows normal activation of the Notch Pathway.

FIG. 2A sets forth an illustration showing the Locations of Epitopes ofInterest on Human ASPH (3D Structure). FIG. 2B lists peptide domainsequences.

FIG. 3 sets forth an illustration showing the Locations of Epitopes ofInterest on the Sequence of ASPH.

FIG. 4 (Panels A-H, plus a polypeptide domain key) sets forth anillustration showing experimentally confirmed and computationallypredicted substrates of ASPH, including those found in the followingtypes of proteins: A. Notch signaling pathway, B. Lipid receptors, C.Blood coagulation cascade proteins, D. Thrombospondins, E. Complementcascade proteins, F. FAT cadherin domain proteins, G. Bone associatedproteins, and H. 7-transmembrane domain containing proteins.

FIG. 5 (Panels A-G, plus a polypeptide domain key) sets forth anillustration showing experimentally confirmed and computationallypredicted substrates of ASPH (continued), including those found in thefollowing types of proteins: A. TGF-b containing proteins, B. Plateletassociated proteins, C. Eye/retina associated proteins, D. Mammarycancer metastasis proteins, E. Slit proteins, F. Miscellaneous proteins,and G. Drosophila homologues.

FIG. 6 sets forth an illustration demonstrating positive 5H4/5K3staining visualized with DAB (brown) on Human Hepatocellular Carcinomaat 4 μg/ml, 8 μg/ml, and 10 μg/ml (3 images). No-primary negativecontrol was performed to identify nonspecific secondary binding (Neg,bottom right image). The scale bar represents 20 μm.

FIG. 7 sets forth an illustration demonstrating positive 9H2/9K1staining visualized with DAB (brown) on Human Hepatocellular Carcinomaat 4 μg/ml, 8 μg/ml, and 10 μg/ml (3 images). No-primary negativecontrol was performed to identify nonspecific secondary binding (Neg,bottom right image). The scale bar represents 20 μm.

FIG. 8 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as LV12 Core F4 (top image), and LV12 CoreF4—Isotype (bottom image). Positive 5H4/5K3 staining was visualized withDAB (brown) on TMAs (LV12 Core F4, top image). Isotype negative controlwas performed with Rabbit IgG (bottom image). The scale bar represents20 μm.

FIG. 9 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as PCO2 Core A6 (top image), and PCO2 CoreA6-Isotype (bottom image). Positive 5H4/5K3 staining was visualized withDAB (brown, top image). Isotype negative control was performed withRabbit IgG (bottom image). The scale bar represents 20 μm.

FIG. 10 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as OV03 Core C5 (top image), and OV03 CoreC5—Isotype (bottom image). Positive 5H4/5K3 staining was visualized withDAB (brown, top image). Isotype negative control was performed withRabbit IgG (bottom image). The scale bar represents 20 μm.

FIG. 11 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as OV01 Core D2 (top image), and OV01 CoreD2—Isotype (bottom image). Positive 5H4/5K3 staining was visualized withDAB (brown, top image). Isotype negative control was performed withRabbit IgG (bottom image). The scale bar represents 20 μm.

FIG. 12 sets forth an illustration demonstrating activity of 5H4/5K3Against Granulosa Cell Tumor Samples (A11 and B11). Positive 5H4/5K3staining was visualized with DAB (brown) Against Granulosa Cell Tumor(top images, A11 and B11) Isotype negative control was performed withRabbit IgG (bottom images, A11 and B11).

FIG. 13 sets forth an illustration demonstrating activity of 5H4/5K3Against Serrous Cystadenocarcinoma Stage III Samples (C5 and D5).Positive 5H4/5K3 staining was visualized with DAB (brown) againstSerrous Cystadenocarcinoma Stage III (top images, C5 and D5). Isotypenegative control was performed with Rabbit IgG (bottom images, C5 andD5).

FIG. 14 sets forth an illustration demonstrating activity of 5H4/5K3Against Serrous Cystadenocarcinoma Stage III Samples (C8 and D8).Positive 5H4/5K3 staining was visualized with DAB (brown) againstSerrous Cystadenocarcinoma Stage III (top images, C8 and D8). Isotypenegative control was performed with Rabbit IgG (bottom images, C8 andD8).

FIG. 15 sets forth an illustration demonstrating activity of 5H4/5K3Against Endometrioid Adenocarcinoma Stage III Samples (E8 and F8).Positive 5H4/5K3 staining was visualized with DAB (brown) againstEndometrioid Adenocarcinoma Stage III (top images, E8 and F8). Isotypenegative control was performed with Rabbit IgG (bottom images, E8 andF8).

FIG. 16 sets forth an illustration demonstrating reaction of 5H4/5K3Against Normal Ovarian Tissue Samples (A1 and B1). Lack of 5H4/5K3staining by DAB against Normal Ovarian Tissue is shown in top images, A1and B1. Isotype negative control was performed with Rabbit IgG (bottomimages, Al and B1).

FIG. 17 sets forth an illustration demonstrating reaction of 5H4/5K3against Thecoma (Theca Cell) Tumor Tissue (A5 and B5). 5H4/5K3 stainingwas visualized with DAB (brown, top images) against Thecoma (Theca Cell)Tumor Tissue (A5 and B5). Isotype negative control was performed withRabbit IgG (bottom images, A5 and B5).

FIG. 18 sets forth an illustration demonstrating Immobilization ofProtein G on Channels 1 (Red, top line) and 2 (Blue, bottom line)followed by Capture of Antibody on Channel 1.

FIG. 19 sets forth an illustration demonstrating Interaction of ASPHwith Mock sample. Concentrations are 500 nM (dark red, top line on rightportion of the graph), 250 nM (light green), 125 nM (blue), 62.5 nM(dark green) 31.2 nM (orange), 15.6 nM (red).

FIG. 20 sets forth an illustration demonstrating Interaction of ASPHwith 2H4/2K5. Concentrations are 500 nM (dark red, 1^(st) line fromtop), 250 nM (light green, 2^(nd) and 3^(rd) lines from top), 125 nM(blue, 4^(th) and 5^(th) lines from top), and 62.5 nM (dark green,6^(th) and 7^(th) lines from top).

FIG. 21 Interaction of ASPH with 5H1/5K1. Concentrations are 500 nM(dark red, 1^(st) line from top), 250 nM (light green, 2^(nd) and 3^(rd)lines from top), 125 nM (blue, 4^(th) and 5^(th) lines from top), 62.5nM (dark green, 6^(th) and 7^(th) lines from top), 31.2 nM (orange,8^(th) and 9^(th) lines from top), and 15.6 nM (red, 10^(th) and 11^(th)lines from top).

FIG. 22 sets forth an illustration demonstrating Interaction of ASPHwith 5H4/5K3. Concentrations are 500 nM (dark red, 1^(st) and 2^(nd)lines from top), 250 nM (light green, 3^(rd) and 4^(th) lines from top),125 nM (blue, 5^(th) and 6^(th) lines from top), 62.5 nM (dark green,7^(th) and 8^(th) lines from top), 31.2 nM (orange, 9^(th) and 10^(th)lines from top), and 15.6 nM (red, 11^(th) and 12^(th) lines from top).

FIG. 23 sets forth an illustration demonstrating Interaction of ASPHwith 9H2/9K1. Concentrations are 500 nM (dark red, 1^(st) and 2^(nd)lines from top), 250 nM (light green, 3^(rd) and 4^(th) lines from top),125 nM (blue, 5^(th) and 6^(th) lines from top), 62.5 nM (dark green,7^(th) and 8^(th) lines from top), 31.2 nM (orange, 9^(th) and 10^(th)lines from top), and 15.6 nM (red, 11^(th) and 12^(th) lines from top).

FIG. 24 sets forth an illustration demonstrating Interaction of ASPHwith 9H2/9K3. Concentrations are 500 nM (dark red, 1^(st) line fromtop), 250 nM (light green, 2^(nd) and 3^(rd) lines from top), 125 nM(blue, 4^(th) and 5^(th) lines from top), 62.5 nM (dark green, 6^(th)line from top), and 31.2 nM (orange, 7^(th) line from top).

FIG. 25 sets forth an illustration demonstrating Interaction of ASPHwith 8H1/8K1. Concentrations are 500 nM (dark red, first and secondlines from the top), 250 nM (light green), 125 nM (blue), 62.5 nM (darkgreen) 31.2 nM (orange), 15.6 nM (red).

FIG. 26 sets forth an illustration demonstrating IC50 Curves for5H4/5K3, 9H2/9K1 and Mock Antibody Samples Carried Out in 4T1 Cells.

FIG. 27 sets forth an illustration demonstrating IC50 Curves for5H4/5K3, 9H2/9K1 and Mock Antibody Samples Carried Out in MCF7 Cells.

FIG. 28 sets forth an illustration demonstrating IC50 Curves for5H4/5K3, 9H2/9K1 and Mock Antibody Samples Carried Out in MV411 Cells.

TABLE #T0 Summary of Staining Patterns in Panels of Photographic Imagesof Cell Samples (from +++ to −)* Top/ Bottom/ Top Top Bottom Bottom FIG.Description Left Right Left Right 6 Positive 5H4/5K3 staining visualizedwith DAB (brown) on + ++ +++ − Human Hepatocellular Carcinoma at 4μg/ml, 8 μg/ml, and 10 μg/ml (3 images). No-primary negative control wasperformed to identify nonspecific secondary binding (Neg, bottom rightimage). 7 Positive 9H2/9K1 staining visualized with DAB (brown) on (+) +++ − Human Hepatocellular Carcinoma at 4 μg/ml, 8 μg/ml, and 10 μg/ml (3images). No-primary negative control was performed to identifynonspecific secondary binding (Neg, bottom right image). 8 5H4/5K3 PhaseIII on TMAs for samples labeled as LV12 Core +++ − F4 (top image), andLV12 Core F4 - Isotype (bottom image). 9 5H4/5K3 Phase III on TMAs forsamples labeled as PC02 Core +++ − A6 (top image), and PC02 Core A6 -Isotype (bottom image). Positive 5H4/5K3 staining was visualized withDAB (brown,, top image). Isotype negative control was performed withRabbit IgG (bottom image). 10 5H4/5K3 Phase III on TMAs for sampleslabeled as OV03 Core +++ − C5 (top image), and OV03 Core C5 - Isotype(bottom image). Positive 5H4/5K3 staining was visualized with DAB(brown, top image). Isotype negative control was performed with RabbitIgG (bottom image). 11 5H4/5K3 Phase III on TMAs for samples labeled asOV01 Core +++ − D2 (top image), and OV01 Core D2 - Isotype (bottomimage). Positive 5H4/5K3 staining was visualized with DAB (brown, topimage). Isotype negative control was performed with Rabbit IgG (bottomimage). 12 Activity of 5H4/5K3 Against Granulosa Cell Tumor Samples ++++ − − (A11 and B11). Positive 5H4/5K3 staining was visualized with DAB(brown) Against Granulosa Cell Tumor (top images, A11 and B11) Isotypenegative control was performed with Rabbit IgG (bottom images, A11 andB11). 13 Activity of 5H4/5K3 Against Serrous Cystadenocarcinoma +++ +++− − Stage III Samples (C5 and D5). Positive 5H4/5K3 staining wasvisualized with DAB (brown) Against Serrous Cystadenocarcinoma Stage III(top images, C5 and D5) Isotype negative control was performed withRabbit IgG(bottom images, C5 and D5). 14 Activity of 5H4/5K3 AgainstSerrous Cystadenocarcinoma +++ +++ − − Stage III Samples (C8 and D8).Positive 5H4/5K3 staining was visualized with DAB (brown) AgainstSerrous Cystadenocarcinoma Stage III (top images, C8 and D8) Isotypenegative control was performed with Rabbit IgG (bottom images, C8 andD8). 15 Activity of 5H4/5K3 Against Endometrioid Adenocarcinoma +++ +++(−) (−) Stage III Samples (E8 and F8). Positive 5H4/5K3 staining wasvisualized with DAB (brown) Against Endometrioid Adenocarcinoma StageIII (top images, E8 and F8). Isotype negative control was performed withRabbit IgG (bottom images, E8 and F8). 16 Reaction of 5H4/5K3 AgainstNormal Ovarian Tissue Samples − − − − (A1 and B1). Lack of 5H4/5K3staining by DAB Against Normal Ovarian Tissue (top images, A1 and B1).Isotype negative control was performed with Rabbit IgG (bottom images,A1 and B1). 17 Reaction of 5H4/5K3 Against Thecoma (Theca Cell) Tumor(+) (+) − − Tissue (A5 and B5). 5H4/5K3 staining was visualized with DAB(brown, top images) Against Thecoma (Theca Cell) Tumor Tissue (A5 andB5). Isotype negative control was performed with Rabbit IgG (bottomimages, AS and B5). *Staining intensities of different panels for eachsample were evaluated on a scale from +++, ++, +, (+), (−), and − wherereaction with DAB to produce an intense brown color after reaction withcells was designated as +++, to −, where all cells were mostly blue orwhite.

TERMS AND DEFINITIONS

The following is a list of abbreviations, plus terms and theirdefinitions, used throughout the specification and the claims:

General abbreviations and their corresponding meanings include: aa orAA=amino acid; mg=milligram(s); ml or mL=milliliter(s);mm=millimeter(s); mM=millimolar; nmol=nanomole(s); pmol=picomole(s);ppm=parts per million; RT=room temperature; U=units; ug, μg=microgram(s); ul, μl=micro liter(s); uM, μM=micromolar.

Specific abbreviations and their corresponding meanings include:

The terms “cell” and “cells,” which are meant to be inclusive, refer toone or more cells which can be in an isolated or cultured state, as in acell line comprising a homogeneous or heterogeneous population of cells,or in a tissue sample, or as part of an organism, such as a transgenicanimal.

The term “amino acid” encompasses both naturally occurring andnon-naturally occurring amino acids unless otherwise designated.

The term “complementarity-determining regions” or “CDRs” are defined byWikipedia, as part of the variable chains in immunoglobulins(antibodies) and T cell receptors, generated by B-cells and T-cellsrespectively, where these molecules bind to their specific antigen.CDRs, which comprise the most variable parts of antibodies, are crucialto the diversity of antigen specificities generated by lymphocytes.

The term “paratope” refers to a set of CDRs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to monoclonal antibodies (MAbs) targetingone or more specific epitopes of aspartyl (asparaginyl) β-hydroxylase(ASPH), including chimeric and humanized MAb variants, and fragmentsthereof (collectively ASPH epitope-specific MAbs, or simply ASPH MAbs),are disclosed. Methods of production, purification, and use of the ASPHepitope-specific MAbs, and compositions comprising them, as agents intherapeutic and diagnostic applications to interact with targetmolecules in cell-free samples, cell- and tissue-based assays, animalmodels, and in a subject are also disclosed. Other aspects of theinvention relate to use of the molecules disclosed herein to diagnose,ameliorate, or treat cell proliferation disorders and related diseases.

One aspect relates to an isolated monoclonal antibody, or a fragmentthereof, which binds to a one or more peptide epitopes of human aspartyl(asparaginyl) β-hydroxylase (ASPH), wherein at least one of said peptideepitopes is located within or adjacent to the catalytic domain of ASPH.

Another aspect relates to an antibody, or a fragment thereof, as notedabove, wherein at least one of said peptide epitopes located within oradjacent to the catalytic domain of ASPH is located within 30 aminoacids of the C-terminus of ASPH.

Another aspect relates to an antibody, which binds to one or moresynthetic peptides selected from the group consisting of (a) a syntheticpeptide comprising 29 amino acids with Cysteine at its amino terminus,plus 28 amino acids corresponding to positions 731-758 at the C-terminalend of human ASPH, with the Threonine at 19 (corresponding to 748 ofASPH) phosphorylated, as CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAIrepresented by SEQ ID NO: 19; and (b) a synthetic peptide comprising 29amino acids with Cysteine at its amino terminus, plus 28 amino acidscorresponding to positions 731-758 at the C-terminal end of human ASPH,as CASSFRLIFIVDVWHPELTPQQRRSLPAI represented by SEQ ID NO: 20.

Related aspects include an antibody, which binds to an epitopecomprising at least 4 consecutive amino acid residues located within 30amino acids from the C-terminal end of human ASPH, including an antibodywherein said epitope comprising at least 4 consecutive amino acidresidues located within 30 amino acids from the C-terminal end of humanASPH comprises the consecutive amino acid selected from the groupconsisting of

PELT represented by SEQ ID NO: 42, ELTP represented by SEQ ID NO: 43,LTPQ represented by SEQ ID NO: 44, TPQQ represented by SEQ ID NO: 45,PQQR represented by SEQ ID NO: 46, QQRR represented by SEQ ID NO: 47,QRRS represented by SEQ ID NO: 48, RRSL represented by SEQ ID NO: 49,RSLP represented by SEQ ID NO: 50, SLPA represented by SEQ ID NO: 51,and LPAI represented by SEQ ID NO: 52.

Related aspects also include an antibody, wherein said peptide epitopecomprises a phosphorylated threonine, T(PO3H2).

Another aspect relates to an isolated monoclonal antibody, or a fragmentthereof, which binds to a one or more peptide epitopes of human aspartyl(asparaginyl) β-hydroxylase (ASPH), wherein said antibody comprises arecombinant heavy chain and a recombinant light chain, wherein saidrecombinant heavy chain comprises a polypeptide sequence selected fromthe group consisting of SEQ ID NOS 21-25; and wherein said recombinantlight chain comprises a polypeptide sequence selected from the groupconsisting of SEQ ID NOS 26-30.

Another aspect relates to an antibody selected from the group consistingof 5H4/5K3 and 9H2/9K1, wherein antibody 5H4/5K3 comprises a heavy chaindesignated 5H4, represented by the sequence SEQ ID NO: 25, and a lightchain 5K3 represented by the sequence SEQ ID NO: 27; and whereinantibody 9H2/9K1 comprises a heavy chain designated 9H2, represented bythe sequence SEQ ID NO: 29, and a light chain 9K1 represented by thesequence SEQ ID NO: 30.

Another aspect relates to an isolated monoclonal antibody, or a fragmentthereof, which binds to a one or more peptide epitopes of human aspartyl(asparaginyl) β-hydroxylase (ASPH), wherein said antibody comprises arecombinant heavy chain comprising:

-   -   a CDR1 comprising a sequence selected from the group consisting        of NFMC (SEQ ID NO: 31), corresponding to residues 50-53 of SEQ        ID NO: 21, and NAMC (SEQ ID NO: 32), corresponding to residues        50-53 of SEQ ID NOS: 23, 29, 24, and 25;    -   a CDR2 comprising a sequence selected from the group consisting        of CIYF (SEQ ID NO: 33) corresponding to residues 68-71 of SEQ        ID NO: 21 and CIDN (SEQ ID NO: 34) corresponding to residues        68-71 of SEQ ID NO: 23, 29, 24, and 25;    -   a CDR3 comprising a sequence selected from the group consisting        of DGPGSISWKI (SEQ ID NO: 35) corresponding to residues 117-126        of SEQ ID NO: 21, and NFNI (SEQ ID NO: 36) corresponding to        residues 116-119 of SEQ ID NOS: 23, 29, 24, and 25.

Another aspect relates to an isolated monoclonal antibody, or a fragmentthereof, which binds to a one or more peptide epitopes of human aspartyl(asparaginyl) β-hydroxylase (ASPH), wherein said antibody comprises arecombinant light chain comprising

-   -   a CDR1 comprising a sequence selected from the group consisting        of SVYSKNR (SEQ ID NO: 37) corresponding to residues 50-56 of        SEQ ID NO: 22, and SVYDNNR SEQ ID NO: 38) corresponding to        residues 50-56 of SEQ ID NOS: 26, 27, 28, and 30,    -   a CDR2 comprising the sequence LAS (SEQ ID NO: 39) corresponding        to residues 78-80 of SEQ ID NOS: 22, 26, 27, 28, and 30;    -   a CDR3 comprising a sequence selected from the group consisting        of QGTYDSSGWYWA (SEQ ID NO: 40) corresponding to residues        113-124 of SEQ ID NO: 22, and [[AND]] LGSYSGYIYI (SEQ ID NO: 41)        corresponding to residues 113-122 of SEQ ID NOS: 26, 27, 28, and        30.

Related aspects include variants of the monoclonal antibodies orfragments thereof, that contain one or more conservative amino acidsubstitutions in which the functional activity relating to binding ofthe antibody or fragment thereof to an epitope of ASPH is retained.Related aspects also include truncated or fusion variants of themonoclonal antibodies comprising one or more insertions or deletions ofamino acids in which the in which the functional activity relating tobinding of the antibody or fragment thereof to an epitope of ASPH isretained. Related aspects also include variants comprising one or morecombinations of conservative amino acid substitutions, insertions, anddeletions, particularly where the number of residues that are altered bysubstitution, insertion, or deletion is small, such as 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, 11-15, 16-20, and 21-25 residues compared to the parentantibody molecule. Related aspects also include molecules having one ormore larger insertions or deletions of amino acid residues orpolypeptide domains that do not alter the functional binding activity ofthe antibody to a desired epitope in a target molecule.

Another aspect relates to a composition comprising any of the antibodiesnoted above, including compositions comprising at least one antibodythat targets ASPH and one or more pharmaceutical excipients.

Another aspect relates to a method of using any of the antibodies notedabove, to inhibit the proliferation of isolated tumor cell samples grownin culture.

Another aspect relates to a method of using any of the antibodies notedabove, to inhibit the proliferation of tumor cells in tissue samplesgrown in culture.

Another aspect relates to a method of treating cancer in a mammaliansubject, comprising administering to a subject in need thereof anantibody as noted above in an amount sufficient to treat cancer. Relatedaspects include methods wherein said mammalian subject is a selectedfrom the group consisting of a human, non-human primate, canine, feline,bovine, equine, and a porcine subject. A preferred aspect relates to amethod, wherein said mammalian subject is a human subject.

Related aspects also include methods noted above wherein said cancer isselected from the group consisting of cancers of the liver,hepatocellular carcinoma and cholangiocarcinoma, pancreatic cancer,gastric cancer, colon cancer, kidney cancer, non-small cell lung cancer,breast cancer, ovarian cancer, cervical cancer, head-and-neck cancerssecondary to human papilloma virus infection, prostate cancer, braincancer, glioblastoma multiform, neuroblastoma, retinoblastoma, andmedulloblastoma, and osteosarcoma.

Another aspect relates to a kit for diagnosis of cancer in a mammaliansubject, wherein said kit comprises an antibody, or a fragment thereof,of any of any of the antibodies noted above.

Another aspect relates to a humanized antibody comprising one or morecomplementarity determining regions (CDRs) derived from a non-humansource targeting one or more peptide epitopes located within or adjacentto the catalytic domain of ASPH of any of claims 1-10, and one or moreportions of the constant regions of a human antibody, and fragmentsthereof.

Another aspect relates to a bispecific antibody comprising one or morecomplementarity determining regions (CDRs) derived from a non-humansource targeting one or more peptide epitopes located within or adjacentto the catalytic domain of ASPH of any of claims 1-10, and an antibodytargeting other epitopes selected from the group consisting of theT-cell redirector class, comprising an antibody targeting one or moreASPH CDRs and an antibody targeting CD3; the NK-cell redirector class,comprising an antibody targeting one or more ASPH CDRs and an antibodytargeting CD16A; the tumor targeting immunomodular class, comprising anantibody targeting one or more ASPH CDRs and an antibody targeting CD40or 4-1BB; and the dual immunomodular class, comprising an antibodytargeting one or more ASPH CDRs and an antibody targeting PD-L1, PD-1,CTLA-4, TGF-β, LAG-3, TIM-3, or OX40.

Therapeutic Uses of Compositions Comprising Compounds of the Invention

Antibodies with direct activity against ASPH antibodies should be usefulin the discovery and development of therapeutic drug products intendedfor use in the treatment of a variety of cancers. These include cancersof the liver, such as hepatocellular carcinoma and cholangiocarcinoma,pancreatic cancer, gastric cancer, colon cancer, kidney cancer,non-small cell lung cancer, breast cancer, ovarian cancer, cervicalcancer, head-and-neck cancers secondary to human papilloma virusinfection, prostate cancer, brain cancers of various types, includingglioblastoma multiform, neuroblastoma, retinoblastoma, andmedulloblastoma, and osteosarcoma.

Pharmaceutical Compositions

Related aspects of the invention are directed to compositions, includingpharmaceutical compositions, comprising the compounds of the invention,noted above. One aspect of the invention is directed to a pharmaceuticalcomposition comprising at least one pharmaceutically acceptableexcipient and a therapeutically effective amount of the compound or saltdisclosed above. Still another aspect of the invention relates to amethod for pharmaceutical formulation of previously described compoundsfor use in oral and intravenous applications, and in implantablematerials.

Another aspect of the present invention relates to a pharmaceuticalcomposition including a pharmaceutically acceptable carrier and acompound according to the aspects of the present invention. Thepharmaceutical composition can contain one or more of theabove-identified compounds of the present invention.

Various Modifications and Alternatives, Generally

While specific aspects of the invention have been described in detail,it will be appreciated by those skilled in the art that variousmodifications and alternatives to those details could be developed inlight of the overall teachings of the disclosure. Accordingly, theparticular arrangements disclosed are meant to be illustrative only, andnot limiting as to the scope of the invention, which is to be given thefull breadth of the appended claims, and any equivalent, thereof.

EXAMPLES

The foregoing discussion may be better understood in connection with thefollowing representative examples which are presented for purposes ofillustrating the principal methods and compositions of the invention,and not by way of limitation. Various other examples will be apparent tothe person skilled in the art after reading the present disclosurewithout departing from the spirit and scope of the invention. It isintended that all such other examples be included within the scope ofthe appended claims.

General Materials and Methods

All parts are by weight (e.g., % w/w), and temperatures are in degreescentigrade (° C.), unless otherwise indicated. Table #T1 presents asummary of the nucleotide and amino acid sequences described in thisapplication.

TABLE#T1 Summary of Sequence IDNumbers Name Description Length TypeSEQ ID NO: Human ASPH Polypeptide corresponding to Human ASPH 758 AA 01deposited as GenBank Accession NoQ12797, starting at the N-terminus withMAQRKNAKSSand ending at the C-terminus with PQQRRSLPAI Canine ASPHPolypeptide corresponding to Canine 798 AA 02ASPH deposited as GenBank Accession No XP_022267901, starting at the N-terminus with MAEETKHGGH and ending at the C-terminus with PQQRHSLPAIPeptide #H1 KRRSNEVLR  corresponding to residues   9 AA 03391-399 of human ASPH Peptide #H2 DRQQFLGHM  corresponding to residues  9 AA 04 428-436 of human ASPH Peptide #H3 GYLLIGDNDN corresponding to residues  10 AA 05 463-470 of human ASPH Peptide #H4RSLYNVNG  corresponding to residues   8 AA 06 562-569 of human ASPHPeptide #H5 PQQRRSLPA I corresponding to residues  10 AA 07749-758 of human ASPH Peptide #H6 FLPEDENLR E corresponding to residues 10 AA 08 612-621 of human ASPH Peptide #H7 VWPHTGPTN Ccorresponding to residues  10 AA 09 676-685 of human ASPH Peptide #H8LWQQGRRN E corresponding to residues   9 AA 10 630-638 of human ASPHPeptide #C1 KRRSNEVLR  corresponding to residues   9 AA 11427-435 of canine ASPH Peptide #C2 DRQQFLGHM  corresponding to residues  9 AA 12 464-472 of canine ASPH Peptide #C3 GYLLIGDNNN corresponding to residues  10 AA 13 499-508 of canine ASPH Peptide #C4RSLYNVHG  corresponding to residues   8 AA 14 598-605 of canine ASPHPeptide #C5 PQQRHSLPA I corresponding to residues  10 AA 15785-794 of canine ASPH Peptide #C6 FLPEDENLR E corresponding to residues 10 AA 16 648-657 of canine ASPH Peptide #C7VWPHIGPINCcorresponding to residues  10 AA 17 712-721 of canine ASPHPeptide #C8 LWQQGRKN E corresponding to residues   9 AA 18666-674 of canine ASPH Peptide #1 Synthetic peptide comprising 29 amino 29 AA 19 (CASSF- acids with Cysteine at its amino PO3H2)terminus, plus 28 amino acids corresponding to positions 731-758 atthe C-terminal end of human ASPH, withthe Threonine at 19 (corresponding to 748 of ASPH) phosphorylated.CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAI Peptide #2Synthetic peptide comprising 29 amino  29 AA 20acids with Cysteine at its amino terminus, plus 28 amino acidscorresponding to positions 731-758 at the C-terminal end of human ASPH.CASSFRLIFIVDVWHPELTPQQRRSLPAI Clone 1H2Translated variable region of Clone ID 150 AA 21#1H2 comprising a GQPK sequence at thestart of the constant region for a heavy chain sequence.METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTASGLSFSDNFMCWVRQAPGKGLEWIACIYFDSSGITYYASWAKGRFTISKTSSPTVTLQMTSLTAADTATYFCARD GPGSISWDLWGQGTLVTVSS GQPKAPSVFPLAP Clone 1K6 Translated variable region of Clone ID 148 AA 22#1K6 comprising a GDPV sequence at thestart of the constant region for a kappa sequence.MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTISCQSSKSVYSKNRLAWYQQKPGQPPKLLIYEASKLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGTYD SSGWYWAFGGGTEVVVK

APTVLIFPPA Clone 5H1 Translated variable region of Clone ID 142 AA 23#5H1. METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGPGHPGHRLERTAESPVGVSTGClone 5H3 Translated variable region of Clone ID 143 AA 24#5H3 comprising a GQPK sequence at thestart of the constant region for a heavy chain sequence.METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGQGTLVTVSS GQPK APSVFPLAPClone 5H4 Translated variable region of Clone ID 143 AA 25#5H4 comprising a GQPK sequence at thestart of the constant region for a heavy chain sequence.METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKASGFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGQGTLVTVSS GQPK APSVFPLAPClone 5K1 1 Translated variable region of Clone 146 AA 26ID#5K1 comprising a GDPV sequence atthe start of the constant region for a kappa sequence.MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK

APTVLIFPPA Clone 5K3 Translated variable region of Clone ID 146 AA 27#5K3 comprising a GDPV sequence at thestart of the constant region for a kappa sequence.MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK

APTVLIFPPA Clone 5K6 Translated variable region of Clone ID 146 AA 28#5K6 comprising a GDPV sequence at thestart of the constant region for a kappa sequence.MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTISCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK

APTVLIFPPA Clone 9H2 Translated variable region of Clone ID 142 AA 29#9H2 comprising a GQPK sequence at thestart of the constant region for a heavy chain sequence.METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKASGFDFISNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNF NLWGQGTL?TVSS GQPK APSVFPLAPClone 9K1 Translated variable region of Clone ID 146 AA 30#9K1 comprising a GDPV sequence at thestart of the constant region for a kappa sequence.MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTISCQSSQSVYDNNRLAWFQQKSGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYS GYIYTFGGGTEVVVK

APTVLIFPPA Clone 1H2 CDR1 region of clone 1H2 corresponding   4 AA 31CDR1 to corresponding to residues 50-53 of SEQ ID NO: 21. NFMCClones 5H1, CDR1 region of Clones 5H1, 9H2, 5H3,   4 AA 32 9H2, 5H3,5H4, corresponding to residues 50-53 of 5H4 CDR1SEQ ID NOS: 23, 29, 24, and 25. NAMC Clone 1H2The CDR2 regions from the heavy chain   4 AA 33 CDR2clone 1H2 corresponding to residues 68-71 of SEQ ID NO: 21. CIYFClones 5H1, The CDR2 regions from the heavy chain   4 AA 34 9H2, 5H3,clones 5H1, 9H2, 5H3, 5H4 corresponding 5H4 CDR2to residues 68-71 of SEQ ID NO: 23, 29, 24, and 25. CIDN Clone 1H2CDR3 regions from the heavy chain clone  10 AA 35 CDR31H2 corresponding to residues 117-126 of SEQ ID NO: 21. DGPGSISWDIClones 5H1, CDR3 regions from the heavy chain   4 AA 36 9H2, 5H3,clones 5H1, 9H2, 5H3, 5H4 corresponding 5H4 CDR3to residues 116-119 of SEQ ID NOS: 23, 29, 24, and 25. NFNI Clone 1K6The CDR1 regions from the kappa chain   7 AA 37 CDR1clone 1K6, corresponding to residues 50-56 of SEQ ID NO: 22. SVYSKNRClones 5K1, The CDR1 regions from the kappa chain   7 AA 38 5K3, 5K6,clones 5K1, 5K3, 5K6, and 9K1 and 9K1corresponding to residues 50-56 of SEQ  CDR1 ID NOS: 26, 27, 28, and 30.SVYDNNR Clones 1K6, The CDR2 regions from the kappa chain   3 AA 395K1, 5K3, clones 1K6, 5K1, 5K3, 5K6, and 9K1 5K6, andcorresponding to residues 78-80 of SEQ  9K1 CDR2ID NOS: 22, 26, 27, 28, and 30. LAS Clone 1K6The CDR3 regions from the kappa chain  12 AA 40 CDR3clone 1K6 corresponding to residues 113-124 of SEQ ID NO: 22.QGTYDSSGWYWA Clones 5K1, The CDR3 regions from the kappa chain  10 AA 415K3, 5K6, clones 5K1, 5K3, 5K6, and 9K1 and 9K1corresponding to residues 113-122 of CDR3SEQ ID NOS: 26, 27, 28, and 30. LGSYSGYIYI PeptideFour aa peptide corresponding to aa   4 AA 42 PELT745-748 near the carboxy terminus of human ASPH. PELT PeptideFour aa peptide corresponding to aa   4 AA 43 ELTP746-749 near the carboxy terminus of human ASPH. ELTP PeptideFour aa peptide corresponding to aa   4 AA 44 LTPQ747-750 near the carboxy terminus of human ASPH. LTPQ PeptideFour aa peptide corresponding to aa   4 AA 45 TPQQ748-751 near the carboxy terminus of human ASPH. TPQQ PeptideFour aa peptide corresponding to aa   4 AA 46 PQRR749-752 near the carboxy terminus of human ASPH. PQQR PeptideFour aa peptide corresponding to aa   4 AA 47 QQRR750-753 near the carboxy terminus of human ASPH. QQRR PeptideFour aa peptide corresponding to aa   4 AA 48 QRSS751-754 near the carboxy terminus of human ASPH. QHRS PeptideFour aa peptide corresponding to aa   4 AA 49 RSSL752-755 near the carboxy terminus of human ASPH. RRSL PeptideFour aa peptide corresponding to aa   4 AA 50 RSLP753-756 near the carboxy terminus of human ASPH. RSLP PeptideFour aa peptide corresponding to aa   4 AA 51 SLPA754-757 near the carboxy terminus of human ASPH. SLPA PeptideFour aa peptide corresponding to aa   4 AA 52 LPAI746-758 near the carboxy terminus of human ASPH. LPAI

Locations of Peptides #H1-#H8 Along Human ASPH (758 aa)ID ASPH_HUMAN              Reviewed;         758 AA.AC Q12797; A0A0A0MSK8; A6NDF4; A6NHI2; B4DIC9; B4E2K4; B7ZM95; E5RGP5;AC F5H667; Q6NXR7; Q8TB28; Q9H291; Q9H2C4; Q9NRI0; Q9NRI1; Q9Y4J0;DT 1 Nov. 1997, integrated into UniProtKB/Swiss-Prot.DT 17 Apr. 2007, sequence version 3. DT 25 Apr. 2018, entry version 181.[...Text omitted...]SQ SEQUENCE 758 AA; 85863 MW; 4AE56D1D8DF0AF0CCRC64;   MAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG HKNGRKGGLS GTSFFTWFMV  60   IALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV DDAKVLLGLK ERSTSEPAVP 120   PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH AEHVEGEDLQ QEDGPTGEPQ 180   QEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ DCNQDMEEMM SEQENPDSSE 240   PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT APPEDNPVED SQVIVEEVSI 300   FPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT IKAELDAAEK LRKRGKIEEA 360                         Peptide #H1<391.399>   VNAFKELVRK YPQSPRARYG KAQCEDDLAE  KRRSNEVLRG AIETYQEVAS LPDVPADLLK 420Peptide #H2<428..436>                 Peptide #H3<463...470>    LSLKRRSDRQ QFLGHM RGSL LTLQRLVQLF PNDTSLKNDL GV GYLLIGDN DN AKKVYEEV 480   LSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP GTDDGRFYFH LGDAMQRVGN 540               Peptide #H4<562569>    KEAYKWYELG HKRGHFASVW Q RSLYNVNGL KAQPWWTPKE TGYTELVKSL ERNWKLIRDE 600    Peptide #H6<612...621>     #H8<630..638>    GLAVMDKAKG L FLPEDENLR EKGDWSQFT L WQQGRRNE NA CKGAPKTCTL LEKFPETTGC 660        Peptide #H7<676...685>    RRGQIKYSIM HPGTH VWPHT GPTNCRLRMH LGLVIPKEGC KIRCANETKT WEEGKVLIFD 720                     Peptide #H5<749...758>   DSFEHEVWQD ASSFRLIFIV DVWHPELT PQ QRRSLPAI  758 //Sequence #5Q2: Locations of Peptides #C1-#C8 AlongCanine ASPH, isoform X1 (794 aa)LOCUS      XP_022267901            794 aa           linear MAM 5 Sep. 2017DEFINITION aspartyl/asparaginyl beta-hydroxylase isoform X1 [Canis lupus           familiaris]. ACCESSION  XP_022267901VERSION    XP_022267901.1 [...Text omitted...] ORIGIN       1 MAEETKHGGH KNGRKGGLSG SSFFTWFMVIA LLGVWTSVA VVWFDLVDYE EVLAKAKDFR      61 YNLSEVLQGK LGVYDADGDG DFDVDDAKVL LGLTKDGSNE NIDSLEEVLN ILAEESSDWF     121 YGFLSFLYDI MTPFEMLEEE EEESETADGV DGLKERSASK PTVPPEEAEP YPWLEEQVIE     181 DSGPQNTEDE VQEVQIESLL HEAVYTEHGD DVQQEEDGQV REPQPEDDFL VGSDTDDRYE     241 PLETGTFHEE TEDSYHIEET ASQAYNQDME EMMYEQDNPD SMEPIVGDDA RTYHEADDLT     301 YQDYDEPVYE PPENEGLESS DNAGEDSNII LEEVYMPPAE EQQEVPPETN RKTDDPEIKE     361 KVKKKKPKLL NKFDKTIKAE LDAAEKLRKR GKIEEALSAF QELVRKYPQS PRARYGKAQC    Peptide #C1<427..435>                    Peptide #C2<464..472>     421 EDDLAE KRRS NEVLR GAIETYQEVASLPNV PTDLLKLTLK RRS DRQQFLG HMRGSLITLQ                  Peptide #C3<499...508>     481 KLVQLFPDDM SLKNDLGV GY LLIGDNNNAQ KVYEEVLNVT PNDGFAKVHY GFILKAQNKI                                                          Peptide #C4<598     541 AESIPYLKEG IESGDPGTDD GRFYFHLGDA MQRVGNKEAY KWYELGHKRG HFASVWQRSL         .605>                                  Peptide #C6<648...657>     601  YNVHG LKAQP WWTPKETGYT ELVKSLERNW KLIRDEGLAV MDKAKGLFLP EDENLRE KGD    Peptide #C8<666..674>                              Peptide #C7<712.....     661 WSQFT LWQQG RKNE NACKGA PKTCSLLDKF PETTGCRRGQ IKYSIMHPGT HVWPHTGPTN      .721>      721  CRLRMHLGLV IPKEGCKIRC ANETKTWEEG KVLIFDDSFE HEVWQDATSF RLIFIVDVWH    Peptide #C5<785...794>      781 PELT PQQRHS LPAI //Sequence #5Q3: Aligned Human ASPH (758 aa) and Canine ASPH,Isoform X1 (794 aa) Sequences Query IDXP_022267901.1Description aspartyl/asparaginyl beta-hydroxylase isoform X1 [Canis lupusfamiliaris] Molecule type amino acid Query Length 794 Subject IDQ12797.3Description RecName: Full = Aspartyl/asparaginyl beta-hydroxylase; AltName:Full = Aspartate beta-hydroxylase; Short = ASP beta-hydroxylase; AltName:Full = Peptide-aspartate beta-dioxygenase Molecule type amino acidSubject Length 758Query   1 MAE-------------------------------ETKHGGHKNGRKGGLSGSSFFTWFMV  29          MA+                               ETKHGGHKNGRKGGLSG+SFFTWFMV Sbjct   1 MAQRKNAKSSGNSSSSGSGSGSTSAGSSSPGARRETKHGGHKNGRKGGLSGTSFFTWFMV  60Query  30 IALLGVWTSVAVVWFDLVDYEEVLAKAKDFRYNLSEVLQGKLGVYDADGDGDFDVDDAKV  89          IALLGVWTSVAVVWFDLVDYEEVL               GKLG+YDADGDGDFDVDDAKV Sbjct  61 IALLGVWTSVAVVWFDLVDYEEVL---------------GKLGIYDADGDGDFDVDDAKV 105Query  90 LLGLTKDGSNENIDSLEEVLNILAEESSDWFYGFLSFLYDIMTPFEMLEEEEEESETADG 149          LLGLSbjct 106 LLGL-------------------------------------------------------- 109Query 150 VDGLKERSASKPTVPPEEAEPYPWLEEQVIEDSGPQNTEDEVQEVQIESLLHEAVYTEH- 208              KERS S+P VPPEEAEP+   EEQV  ++ PQN EDE +E QI+SLLHE V+ EHSbjct 110 ----KERSTSEPAVPPEEAEPHTEPEEQVPVEAEPQNIEDEAKE-QIQSLLHEMVHAEHV 164Query 209 -GDDVQQEEDGQVREPQPEDD-FLVGSDTDDRYEPLETGTFHEETEDSYHIEETASQAYN 266           G+D+QQE DG   EPQ EDD FL+ +D DDR+E LE    HEETE SYH+EET SQ  NSbjct 165 EGEDLQQE-DGPTGEPQQEDDEFLMATDVDDRFETLEPEVSHEETEHSYHVEETVSQDCN 223Query 267 QDMEEMMYEQDNPDSMEPIVGDDARTYHEADDLTYQDYDEP-VYEPPENEGLESS----- 320          QDMEEMM EQ+NPDS EP+V +D R +H+ DD+TYQ Y+E  VYEP ENEG+E +Sbjct 224 QDMEEMMSEQENPDSSEPVV-EDERLHHDTDDVTYQVYEEQAVYEPLENEGIEITEVTAP 282Query 321 --DNAGEDSNIILEEVYMPPAEEQQEVPPETNRKTDDPEIKEKVKKKKPKLLNKFDKTIK 378            DN  EDS +I+EEV + P EEQQEVPPETNRKTDDPE K KVKKKKPKLLNKFDKTIKSbjct 283 PEDNPVEDSQVIVEEVSIFPVEEQQEVPPETNRKTDDPEQKAKVKKKKPKLLNKFDKTIK 342                                               Peptide #C1<427.435>Query 379 AELDAAEKLRKRGKIEEALSAFQELVRKYPQSPRARYGKAQCEDDLAE KRRSNEVLRGAI 438          AELDAAEKLRKRGKIEEA++AF+ELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAISbjct 343 AELDAAEKLRKRGKIEEAVNAFKELVRKYPQSPRARYGKAQCEDDLAE KRRSNEVLRGAI 402                                               Peptide #H1<391.399>                        Peptide #C2<464.472>Query 439 ETYQEVASLPNVPTDLLKLTLKRRS DRQQFLGHMRGSLITLQKLVQLFPDDMSLKNDLGV 498          ETYQEVASLP+VP DLLKL+LKRRSDRQQFLGHMRGSL+TLQ+LVQLFP+D SLKNDLGVSbjct 403 ETYQEVASLPDVPADLLKLSLKRRS DRQQFLGHMRGSLLTLQRLVQLFPNDTSLKNDLGV 462                        Peptide #H2<428..436> Peptide #C3<499..508>Query 499  GYLLIGDNNNAQKVYEEVLNVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT 558          GYLLIGDN + NA+KVYEEVL+VTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT                  * Sbjct 463  GYLLIGDNDNAKKVYEEVLSVTPNDGFAKVHYGFILKAQNKIAESIPYLKEGIESGDPGT 522 Peptide #H3<463..470>                                      Peptide #C4<598605>Query 559 DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQ RSLYNVHGLKAQPWWTPKETG 618          DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQRSLYNV + GLKAQPWWTPKETG                                                       *Sbjct 523 DDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQ RSLYNVNGLKAQPWWTPKETG 582                                      Peptide #H4<562569>                            Peptide #C6<648..657>     #C8<666.674>Query 619 YTELVKSLERNWKLIRDEGLAVMDKAKGL FLPEDENLRE KGDWSQFT LWQQGRKNENACK 678          YTELVKSLERNWKLIRDEGLAVMDKAKGLFLPEDENLREKGDWSQFTLWQQGR + NENACK                                                               *Sbjct 583 YTELVKSLERNWKLIRDEGLAVMDKAKGL FLPEDENLRE KGDWSQFT LWQQGRRNENACK 642                             Peptide #H6<612..621>    #H8<630.638>                                Peptide #C7<712..721>Query 679 GAPKTCSLLDKFPETTGCRRGQIKYSIMHPGTH VWPHTGPTNCRLRMHLGLVIPKEGCKI 738          GAPKTC+LL+KFPETTGCRRGQIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPKEGCKISbjct 643 GAPKTCTLLEKFPETTGCRRGQIKYSIMHPGTH VWPHTGPTNCRLRMHLGLVIPKEGCKI 702                                Peptide #H7<676-685>                                             Peptide #C5<785..794>Query 739 RCANETKTWEEGKVLIFDDSFEHEVWQDATSFRLIFIVDVWHPELT PQQRHSLPAI     794          RCANETKTWEEGKVLIFDDSFEHEVWQDA+SFRLIFIVDVWHPELTPQQR_SLPAI                                                            *Sbjct 703 RCANETKTWEEGKVLIFDDSFEHEVWQDASSFRLIFIVDVWHPELT PQQRRSLPAI     758                                             Peptide #H5<749..758>

Example 1—Design and Synthesis of Synthetic Peptides Corresponding toEpitopes of ASPH Synthesis of Exemplary Compounds

Synthetic peptides derived from human and/or canine ASPH were designedthat correspond to eight domain regions (#1-#8, as #H1-#H8 and #C1-#C8),as penultimate domain epitopes of the full length polypeptide, asillustrated in FIG. 2A, and shown in FIG. 2B and below in Table #T2.These peptides were rationally selected based upon the spatial distancefrom the substrate, as found in crystal structure 5JZZ deposited at theRCSB Protein Databank. Peptide epitope domain regions #1-#3 are from thenon-catalytic domain, while peptide epitope domain regions #4 & #5 arefrom the C-terminal catalytic domain, but outside of residues 650-700.Peptide epitope domain regions #6, #7, and #8 are within or near theC-terminal catalytic domain of ASPH.

TABLE #T2 Peptide Sequences Corresponding to PenultimateDomain Epitopes of Human and Canine ASPH Positions Positions EpitopeShort Human in Canine SEQ Domain Organism Sequence Name ASPH ASPH ID NOS#1 HUMAN/CANINE KRRSNEVLR #H1/#C1 391-399 427-435 03/11 #2 HUMAN/CANINEDEQQFLGHM #H2/C2 428-436 464-472 04/12 #3 HUMAN GYLLIGDNDN #H3 463-47005 CANINE GYLLIGDNNN #C3 499-508 13 #4 HUMAN RSLYNVNG #H4 562-569 06CANINE RSLYNVHG #C4 598-605 14 #5 HUMAN PQQRRSLPAI #H5 749-758 07 CANINEPQQRHSLPAI #C5 785-794 15 #6 HUMAN/CANINE FLPEDENLRE #H6/C6 612-621648-657 08/16 #7 HUMAN/CANINE VWPHTGPTNC #H7/C7 676-685 712-721 09/17 #8HUMAN LWQQGRRNE #H8 630-638 10 CANINE LWQQGRKNE #C8 666-674 18

Example 2—Immunization of Peptide Candidates into Rabbits and Test Bleed

ImmunoPrecise Antibodies Ltd. (Victoria, British Columbia, Canada)carried out immunization of peptide candidates into rabbits, the testingof antibodies from rabbit B cells, cloning of variable regions intoexpression vectors, and DNA sequencing of selected rabbit MAbs (Examples2-8) using standard procedures, under contract with principalinvestigators at Midwestern University (Glendale, Ariz.).

TABLE #T3Synthetic Peptide Sequences Used as Immunogens Directed against ASPHPositions Short in Human Epitope SEQ Name Description/Sequence ASPHDomain ID NOS Peptide Synthetic peptide comprising 29 amino 731-758 #519 (CASSF- acids with Cysteine at its amino terminus, PO3H2)plus 28 amino acids corresponding topositions 731-758 at the C-terminal end ofhuman ASPH, with the Threonine at 19 (corresponding to 748 of ASPH)phosphorylated. C-ASSFRLIFIV DVWHPEL-T(PO3H2)-PQ QRRSLPAI PeptideSynthetic peptide comprising 29 amino 731-758 #5 20acids with Cysteine at its amino terminus,plus 28 amino acids corresponding topositions 731-758 at the C-terminal end of human ASPH.C-ASSFRLIFIV DVWHPELTPQ QRRSLPAI

Synthetic peptides #1 and #2 (1 mg each) were prepared at a purityof >95%. The N-terminal Cysteine residue on each peptide is used tofacilitate conjugation of each peptide to other molecules. BSA and KLH(2 mg each) were synthesized or obtained from commercial sources.

Peptide #1 (SEQ ID NO: 19) CASSFRLIFIVDVWHPEL-T(PO3H2)-PQQRRSLPAIPeptide #2 (SEQ ID NO: 20) CASSFRLIFIVDVWHPELTPQQRRSLPAI

Briefly, 3-6 mg of immunizing/screening antigen were prepared and storedin a neutral pH, sterile, buffered solution, at a minimum concentrationof 0.5 mg/L. Antigen (hapten) was conjugated to an appropriate carrierand emulsified in Freund's Complete adjuvant, and used to immunize twoNew Zealand White (NZW) rabbits by subcutaneous injections. Boosterinjections of antigen in Freund's Incomplete adjuvant were carried at 3week intervals. Blood samples (test bleeds) were collected 7-10 daysafter the second boost and immune sera were tested for specific antibodytiter by ELISA. Each rabbit was given a final boost, if required, andwhole blood was used to obtain B cells to generate Monoclonal Antibodies(MAbs) by the methods noted below.

Example 3—In Vitro Culture of Rabbit B Cells

Whole rabbit blood was collected after the final boost, and B cells wereisolated, purified, and cultured by ImmunoPrecise Antibodies Ltd.

Example 4—Screening and Analysis of Antibodies from Rabbit B Cells

Screening was performed on the immunizing antigen by an indirect ELISAperformed by ImmunoPrecise Antibodies Ltd.

ELISA plates were obtained from Costar Corning (Catalog #0720039).Blocking solutions included BSA (Bovine serum albumin) and Skim milkpowder (MP). Phosphate buffered saline (PBS) at pH 7.4, PBS with 0.05%Tween-20 at pH 7.4, and Carbonate coating buffer (CCB) at pH 9.6 wereused in the ELISA tests. Primary antibodies being tested included theimmune sera, B cell supernatants, and transfected supernatants(recombinant rabbit MAbs). Secondary antibodies included GoatAnti-Rabbit IgG-Fc-HRP, Subisotype IgG1, obtained from JacksonImmunoResearch (Catalog #111-035-046), and AffiniPure goat anti-rabbitIgG (H+L), Subisotype IgG1, obtained from Jackson ImmunoResearch(Catalog #111-035-144). Substrate reagents included TMB (3,3′,5,5′-tetramethyl-benzidine buffer), TMB One Component HRP Microwell Substrate, andBioFx cat #TMBW-1000-01.

Briefly, B cell culture supernatants from 96-well plates weretransferred to ELISA plates coated with antigen. An indirect ELISA wasperformed by probing each well with a secondary antibody that binds torabbit IgG antibodies. Wells with cells that tested positive wereretested with the immunizing antigen to confirm specificity and binding.

Samples corresponding to the top responding wells were preserved inlysis buffer.

Cell culture supernatants from positive wells (in a volume of <50 μL)were also preserved.

Example 5—Cloning Antibody Heavy and Light Chain Variable Regions inMammalian Expression Vectors

Cells from selected wells of B cells were amplified and samples of mRNAprepared from those cells by ImmunoPrecise Antibodies Ltd. ComplementaryDNAs corresponding to rabbit IgG heavy and kappa light chain variableregions were prepared and cloned separately into mammalian expressionvectors comprising rabbit heavy and light chain constant regions,respectively.

Example 6—Expression of Antibody Heavy and Light Chain Variable Regionsinto HEK293 Cells

Two plasmids, one comprising a heavy chain variable and a constantregion and one comprising a light chain variable and constant region,were co-transfected into HEK293 cells, and allowed to express bothchains of the rabbit antibodies.

Example 7—Analysis of Cell Culture Supernatants

The cell culture supernatants were assayed for activity by indirectELISA against the immunizing peptide (Peptide #1, SEQ ID NO: 13). Tenclones (#1-#10) having positive activity against immunizing peptide wereidentified. One clone produced an antibody that reacted with thephosphorylated Peptide #1, and four clones produced antibodies thatreacted against both the phosphorylated Peptide #1 (SEQ ID NO: 19) andthe non-phosphorylated Peptide #2 (SEQ ID NO: 20).

Example 8—DNA Sequencing of Heavy and Light Chain Regions from SelectedPositive Rabbit MAbs

Ten clones were selected, five comprising heavy chains (1H2, 5H1, 5H3,5H4 and 9H2), and five comprising kappa chains (1K6, 5K1, 5K3, 5K6 and9K1). Purified plasmid DNA samples were prepared and sent to MacrogenUSA for sequencing and analyzed by SnapGene Version 4.0.4.

The rabbit IgG heavy chain sequence is about 1200 bp in length, and canbe sequenced from its 5′ end to obtain a reliable full-length variablesequence. The rabbit kappa light chain is about 700 bp in length, andfull-length variable sequence can be reliably obtained from sequencingin the 5′ direction.

Analysis of Translation of Consensus Amino Acid Sequences

The nucleotide sequences of the variable regions of five heavy chainsand five kappa chains were analyzed. Table #T4 discloses the translatedvariable regions encoded by the nucleotide sequences of the top 10clones. Sequences highlighted in bold with a single underline (as GQPK)show the start of the constant region for heavy chains, and sequenceshighlighted in italic and double underline (as GDPV) show the start ofthe constant region of kappa chains.

TABLE #T4 Translated variable region sequences of the top clones #Clone ID Description and Sequence Length Type SEQ ID NO  1 1H2METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCTAS 250 AA 21GLSFSDNFMCWVRQAPGKGLEWIACIYFDSSGITYYASWAKGRFTISKTSSPTVTLQMTSLTAADTATYFCARDGPGSISWDLWGQ GTLVTVSS GQPK APSVFPLAP  21K6 MDTRAPTQLLGLLLLWLPGATFAQVLTQTPSPVSAAVGGTVTI 148 AA 22SCQSSKSVYSKNRLAWYQQKPGQPPKLLIYEASKLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGTYDSSGWYWAFGGGT EVVVK

APTVLIFPPA  3 5H1 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 142 AA 23GFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGPGHPGHRL ERTAESPVGVSTG  4 5H3METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 143 AA 24GFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTLVTVS S GQPK APSVFPLAP  5 5H4METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 143 AA 25GFDFSSNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTLVTVS S GQPK APSVFPLAP  6 5K1MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 26SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK

APTVLIFPPA  7 5K3 MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 27SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK

APTVLIFPPA  8 5K6 MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 28SCQSSQSVYDNNRLAWFQQKPGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK

APTVLIFPPA  9 9H2 METGLRWLLLVAVLKGVQCQSLEESGGDLVKPGASLTLTCKAS 142 AA 29GFDFISNAMCWVRQAPGKGPEWIACIDNGDGSTDYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCTRNFNLWGQGTL?TVS S GQPK APSVFPLAP 10 9K1MDTRAPTQLLGLLLLWLPGATFAQVLTQTASSVSAAVGGTVTI 146 AA 30SCQSSQSVYDNNRLAWFQQKSGQPPKLLIYETSKLASGVPLRFKGSGSGTQFTLTISDLECDDAATYYCLGSYSGYIYTFGGGTEV VVK

APTVLIFPPA

These results demonstrate that recombinant monoclonal antibodies derivedfrom rabbits, were generated successfully against Peptide #1 (SEQ ID NO:13). Recombinant Clones 5H1, 5H3, 5H4, and 9H2 have the same heavy chainsequences, and recombinant clones 5K1, 5K3 and 9K1 have the same kappachain sequence.

Sequence #SQ4: Multiple Sequence Alignment of Heavy Chains for Clones1H2, 5H1, 9H2, 5H3, and 5H4

A multiple sequence alignment of five clones comprising heavy chainsillustrates slight differences in the encoded polypeptide sequences inregions within and just flanking CDR1, CDR2, CDR3, with notabledivergence for sequences after CDR3 for clone 5H1.

CLUSTAL O(1.2.4) multiple sequence alignment heavy chains:

The CDR1 regions from the heavy chain clones include the sequences NFMC(SEQ ID NO: 31), corresponding to residues 50-53 of SEQ ID NO: 21, andNAMC (SEQ ID NO: 32), corresponding to residues 50-53 of SEQ ID NOS: 23,29, 24, and 25. The CDR2 regions from the heavy chain clones includeCIYF (SEQ ID NO: 33) corresponding to residues 68-71 of SEQ ID NO: 21,and CIDN (SEQ ID NO: 34) corresponding to residues 68-71 of SEQ ID NO:23, 29, 24, and 25. The CDR3 regions from the heavy chain clones includeDGPGSISWDI (SEQ ID NO: 35) corresponding to residues 117-126 of SEQ IDNO: 21, and NFNI (SEQ ID NO: 36) corresponding to residues 116-119 ofSEQ ID NOS: 23, 29, 24, and 25.

Sequence #SQ5: Multiple Sequence Alignment of Kappa Light Chains forClones 1K6, 5K1, 5K3, 5K6, and 9K1

A multiple sequence alignment of five clones comprising kappa lightchains illustrates slight differences in the encoded polypeptidesequences in regions within and just flanking CDR1, CDR2, CDR3, withnotable divergence for sequences within CDR3 for clone 1K6.

CLUSTAL O(1.2.4) multiple sequence alignment kappa chains:

The CDR1 regions from the kappa chain clones 1K6, 5K1, 5K3, 5K6, and 9K1include SVYSKNR (SEQ ID NO: 37) corresponding to residues 50-56 of SEQID NO: 22, and SVYDNNR (SEQ ID NO: 38) corresponding to residues 50-56of SEQ ID NOS: 26, 27, 28, and 30. The CDR2 regions from the kappa chainclones were all LAS (SEQ ID NO: 39) corresponding to residues 78-80 ofSEQ ID NOS: 22, 26, 27, 28, and 30. The CDR3 regions from the kappachain clones included QGTYDSSGWYWA (SEQ ID NO: 40) corresponding toresidues 113-124 of SEQ ID NO: 22, and LGSYSGYIYI (SEQ ID NO: 41)corresponding to residues 113-122 of SEQ ID NOS: 26, 27, 28, and 30.

Example 9—Analysis of MAbs by Immunohistochemistry (IHC)—PhaseI—Antibody Triage

Antibody triage (Phase I) was performed by Reveal Biosciences (SanDiego, Calif.) on a Leica Bond automated immunostainer, testing eachantibody at 8 μg/mL, in parallel with a negative control performed inabsence of primary antibody. FFPE human hepatocellular carcinoma wasused for antibody testing.

Heat induced antigen retrieval was performed using Leica Bond EpitopeRetrieval Buffer 1 (Citrate Buffer, pH6.0) and Leica Bond EpitopeRetrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)).Non-specific antibody binding was blocked using 3% Normal Goat Serum inPBST. Tests for positive reactions were carried out by using NovocastraBond Refine Polymer Detection reagent, and visualized with3′3-diaminobenzidine (DAB; brown). A Hematoxylin nuclear counterstain(blue) was also applied.

When Phase I optimization slides were evaluated, only two samples,5H4/5K3 and 9H2/9K1, showed positive staining in Epitope RetrievalBuffer, ER2(20), as noted below.

TABLE #T5 Results of Antibody Triage Groups Antibody Dilution HostSpecies Antigen Retrieval 1 1H2/1K6 NONE 1H2/1K5 NONE 1H4/1K6 NONE1H4/1K4 NONE 8 μg/mL Rabbit 2 2H4/2K5 NONE 5H1/5K1 NONE 5H4/5K3 ER2(20)9H2/9K1 ER2(20)

Two antibodies, 5H4/5K3 and 9H2/9K1, that showed positive staining inER2(20), were selected for further testing in Phase II.

Example 10A—Analysis of MAbs by Immunohistochemistry (IHC)—Phase II—IHCOptimization

Immunohistochemistry (IHC) Optimization was performed by RevealBiosciences (San Diego, Calif.) on a Leica Bond automated immunostainer,by testing each antibody at 2 μg/mL, 4 μg/mL, 8 μg/mL, and 10 μg/mL.

Heat induced antigen retrieval was performed using Leica Bond EpitopeRetrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)).Non-specific antibody binding was blocked using 3% Normal Goat Serum inPBST. Tests for positive reactions were carried out by using NovocastraBond Refine Polymer Detection and visualized with 3′3-diaminobenzidine(DAB; brown). A Hematoxylin nuclear counterstain (blue) was applied.

When Phase II optimization samples were evaluated, no staining wasobserved at 2 μg/mL for 5H4/5K3 and 9H2/9K1. A strong signal wasdetected at both 8 μg/mL and 10 μg/mL for 5H4/5K3, as illustrated inFIG. 6. A strong signal was detected at both 8 μg/mL, and 10 μg/mL for9H2/9K1, with a stronger intensity at 10 μg/mL, as illustrated in FIG.7.

These results demonstrate that 5H4/5K3 and 9H2/9K1 are notable as leadsfor the development of diagnostic agents, and also as therapeutic drugproducts suitable for use in mammals, such as humans, by grafting theCDRs onto a suitable antibody framework that will facilitate thetargeting of one or more drug products to cancerous tissues in a humansubject.

Example 10B—Analysis of MAbs by Immunohistochemistry (IHC)—Phase III—IHCon Tissue Micro Arrays

Immunohistochemistry (IHC) was performed on a Leica Bond automatedimmunostainer using 5H4/5K3 at 8 μg/mL on TMAs (Table #T5).

Heat induced antigen retrieval was performed using Leica Bond EpitopeRetrieval Buffer 2 (EDTA solution, pH9.0) for 20 minutes (ER2(20)).Non-specific antibody binding was blocked using 3% Normal Goat Serum inPBST.

Positivity was detected using Novocastra Bond Refine Polymer Detectionand visualized with 3′3-diaminobenzidine (DAB; brown). A Hematoxylinnuclear counterstain (blue) was applied.

Isotype controls were performed on Human Hepatocellular carcinoma slideand each TMA type alongside their respective positive (with primary)slide using Rabbit IgG (Abcam ab172730, lot #GR3179509-3).

A human hepatocellular carcinoma FFPE block was sectioned at 4 umthickness and mounted onto positively charged slides for assaydevelopment.

TABLE #T6 Tissue Micro Arrays used for IHC staining in Phase III ArrayName Tissue Type LV12 Liver cancer tissue array with progressive changesNT01 Normal Human Tissue PC02 Pancreatic cancer tissue array OV01 Ovarycancer tissue array OV03 Ovary cancer tissue array with progressivechanges

FIG. 8 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as LV12 Core F4 (top panel), and LV12 CoreF4—Isotype (bottom panel). Positive 5H4/5K3 staining was visualized withDAB (brown). Isotype negative control was performed with Rabbit IgG(right images). The scale bar represents 20 μm.

FIG. 9 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as PC02 Core A6 (top panel), and PC02 CoreA6—Isotype (bottom panel).

FIG. 10 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as OV03 Core C5 (top panel), and OV03 CoreC5—Isotype (bottom panel).

FIG. 11 sets forth an illustration demonstrating 5H4/5K3 Phase III onTMAs for samples labeled as OV01 Core D2 (top panel), and OV01 CoreD2—Isotype (bottom panel).

These results demonstrate that antibody 5H4/5K3 stains a broad range ofovarian cancer samples, from granuloma to serous to endometrioidcancers. Malignant cancers stain intensely, while benign and normalovarian tissue samples do not stain under these conditions.

These and similar antibodies, plus fragments or derivatives thereof,should be useful as a key reagent in a kit to diagnose the presence ofcancer cells in wide variety of research and clinical samples.

These and similar antibodies, plus fragments or derivatives thereof, mayalso be useful in the development of pharmaceutical compositionscomprising a therapeutic agent when the CDRs are grafted onto anappropriate framework suitable to produce a drug product suitable formammals, particularly non-human primate and human subjects, andlivestock, and domestic pets, including dogs and cats.

Example 10C—Analysis of MAbs by Immunohistochemistry (IHC)—Phase III—IHCon Tissue Micro Arrays

FIG. 12 sets forth an illustration demonstrating activity of 5H4/5K3Against Granulosa Cell Tumor Samples (A11 and B11). Positive 5H4/5K3staining was visualized with DAB (brown) against Granulosa Cell Tumor(top images, A11 and B11). Isotype negative control was performed withRabbit IgG (bottom images, A11 and B11).

FIG. 13 sets forth an illustration demonstrating activity of 5H4/5K3Against Serrous Cystadenocarcinoma Stage III Samples (C5 and D5).

FIG. 14 sets forth an illustration demonstrating activity of 5H4/5K3Against Serrous Cystadenocarcinoma Stage III Samples (C8 and D8).

FIG. 15 sets forth an illustration demonstrating activity of 5H4/5K3Against Endometrioid Adenocarcinoma Stage III Samples (E8 and F8).

FIG. 16 sets forth an illustration demonstrating reaction of 5H4/5K3Against Normal Ovarian Tissue Samples (A1 and B1).

FIG. 17 sets forth an illustration demonstrating reaction of 5H4/5K3Against Thecoma (Theca Cell) Tumor Tissue (A5 and B5).

These results confirm activity of the 5H4/5K3 antibody against a varietyof cancerous tissue samples, and a lack of activity against cells innormal tissue samples.

Example 11—Interactions Between ASPH and Selected MAbs Captured ViaProtein G

The interaction between ASPH and a set of 6 antibodies werecharacterized by Essai Sciences LLC (Stillwater, Okla.) on a SensiQPioneer SPR Platform. The COOH2 sensor chip, which contains a planardextran surface, was used for target immobilization. The buffer systemwas 10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.01% Tween-20.

All channels of a COOH2 sensor chip were activated with a five-minuteinjection of 40 mM EDC and 10 mM NHS. Protein G was then injected acrosschannels 1 and 2. 1 M ethanolamine, pH 8.0 was then injected across allthree channels. Approximately 1000 response units of Protein G werecaptured on both channels 1 and 2 (FIG. 18). For each antibody-ASPHinteraction, the antibody was injected on channel 1, leaving channels 2and 3 as a Protein G reference and empty channel reference,respectively. After antibody capture, ASPH was injected at a singleconcentration. Following injection of ASPH, all three channels wereinjected with 10 mM NaOH for one minute to regenerate the Protein Gsurface. This was done twice for each antibody, at each testedconcentration of ASPH.

All experimental results shown are from fixed-concentration analyses ofthe interactions. Given material constraints, as well as the nature ofthe interacting molecules, immobilization of the antibodies, andfixed-concentration injection of ASPH was the most feasible experimentalsetup for this study.

The response curves for each tested concentration of ASPH against eachcaptured antibody are displayed below. FIG. 19 is the mock sample, whichdemonstrates no visible binding. The remaining antibodies (FIGS. 20-25)display affinity for ASPH that range from ^(˜)60 nM (9H2/9K3, FIG. 24)to 920 nM (2H4/2K5, FIG. 22). We tested the phospho-selective antibody,8H1/8K1 (FIG. 25), and observed no binding, even at the highest testedanalyte concentration. The kinetics values for each interaction arelisted in Table #T7.

TABLE #T7 Kinetics values for interaction of ASPH with antibodies.Antibody ka (M⁻¹s⁻¹) kd (s⁻¹) K_(D) (M) Mock — — — 2H4/2K5  1.83 ±0.04e3 1.681 ± 0.002e−3   920 ± 20 nM 5H1/5K1 2.035 ± 0.002e4 2.410 ±0.002e−3 118.4 ± 0.2 nM 5H4/5K3 1.683 ± 0.002e4 2.426 ± 0.002e−3 144.2 ±0.2 nM 9H2/9K1 1.879 ± 0.002e4 2.363 ± 0.002e−3 125.7 ± 0.2 nM 9H2/9K32.985 ± 0.004e4 1.848 ± 0.003e−3  61.9 ± 0.1 nM 8H1/8K1 — — —

The interaction of the ASPH protein with a set of antibodies capturedvia Protein G was studied. A range of affinities from ^(˜)60 nM to^(˜)920 nM for the binding antibodies was observed. A mock sample, and aphospho-selective antibody were also tested. No observable binding tothe protein for the mock sample or the phospho-selective antibody wasnoted.

Example 12—In Vitro Cell Proliferation Assay with Antibodies AgainstEpitopes of ASPH in Three Tumor Cell Lines

Experiments to determine the half maximal inhibitory concentration(IC₅₀) of the potency of samples comprising selected antibodies indifferent types of cultured tumor cells were carried out byTranslational Drug Development LLC.

FIG. 26 shows graphs illustrating IC₅₀ curves for three samples testedin 4T1 Murine Breast Tumor cells (Panel A, 5H4/5K3; Panel B, 9H2/9K1;and Panel C, Mock Antibody).

FIG. 27 shows graphs illustrating IC₅₀ curves for three samples testedin MCF-7 Human ER+Breast Tumor cells (Panel A, 5H4/5K3; Panel B,9H2/9K1; and Panel C, Mock Antibody).

FIG. 28 shows graphs illustrating IC₅₀ curves for three samples testedin MV411 Human Mantle Cell Leukemia cells (Panel A, 5H4/5K3; Panel B,9H2/9K1; and Panel C, Mock Antibody).

TABLE #T8 Summary of IC₅₀ Results* Mean IC₅₀ Mean IC₅₀ Mean IC₅₀ (μg/mL)(μg/mL) (μg/mL) Mock Cell Line Tissue Type 5H4/5K3 9H2/9K1 Antibody 4T1Murine Breast Tumor 0.026 0.008 0.280 MCF-7 Human ER+ Breast Tumor 0.0240.002 0.426 MV411 Human Mantle Cell 0.098 0.007 0.313 Leukemia *Mean ICvalues are calculated as the average of IC₅₀ values obtained from twotrials, A and B, for each of 3 antibody experiments in 3 cell lines, asnoted in Panels A-C of FIGS. 26 through 28.

These results demonstrate that the antibodies designated as 5H4/5K3 and9H2/9K1 both affect the viability of three tumor cell lines beingtested, with the Mab designated 9H2/9K1 being more potent than the Mabdesignated 5H4/5K3.

The antibody designated as 5H4/5K3 appears to be more selective forbreast tumors 4T1 and MCF-7.

Example 13—Generation of Humanized Chimeric Monoclonal AntibodiesTargeting at Least One Epitope in the Catalytic Domain of ASPH

Humanized versions of non-human antibodies are chimeric antibodies thata minimal amount of polypeptide domains comprising amino acid sequencesderived from the non-human antibody. Typically, residues from thehypervariable region of a human antibody are replaced with hypervariableresidues from the non-human antibody that have the desired specificity,affinity, and/or capacity. Humanized versions can also be prepared fromnon-human species, such as mouse, rat, rabbit, non-human primates, andother vertebrate species. Other regions, comprising amino acid residuesthat may contribute to structural integrity of the human antibody(framework region) may also be replaced by amino acid residues from thecorresponding non-human residues. The humanized chimeric monoclonalantibodies may also comprise amino acid residues that are not found inthe recipient human antibody or the non-human donor antibody. Generally,the humanized antibody comprises at least one, and preferably all of thevariable domains of the donor antibody, and substantially all of theframework regions of the human antibody.

Variants may also comprise one or more portions of the constant regionof an antibody, typically, a human antibody. Other types of variants,include fragments, and variants comprising one or more conservativesubstitutions, insertions, or deletions, that do not substantially alterthe specificity, affinity, and/or capacity of the variant moleculecompared to its parent molecule, but may offer additional advantages interms of ease of production or purification, ability to be conjugated toother chemical moieties, which may facilitate covalent or non-covalentbinding to other molecules comprising polypeptide domains or otherreactive or non-reactive moieties, capable of providing a secondaryreporter function, such as emission of fluorescent light, or conversionof a colorless substrate to an easily detectable, colored product, whichmay be useful as components in diagnostic kits for use in research andin clinical settings. Aspects of the invention also include variantsthatare >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >98%, >99%,or >99.5% identical to at least one of the variable regions of the donorantibody.

In the examples noted above, recombinant monoclonal antibodies weregenerated against Peptide #1 (SEQ ID NO: 13). Recombinant Clones 5H1,5H3, 5H4, and 9H2 have the same heavy chain sequences, and recombinantclones 5K1, 5K3 and 9K1 have the same kappa chain sequence. The CDR1regions from the heavy chain clones include the sequences NFMC (SEQ IDNO: 31) and NAMC (SEQ ID NO: 32). The CDR2 regions from the heavy chainclones include CIYF (SEQ ID NO: 33) and CIDN (SEQ ID NO: 34). The CDR3regions from the heavy chain clones include DGPGSISWDI (SEQ ID NO: 35)and NFNI (SEQ ID NO: 36). The CDR1 regions from the kappa chain clonesinclude SVYSKNR (SEQ ID NO: 37) and SVYDNNR(SEQ ID NO: 38). The CDR2regions from the kappa chain clones were all LAS (SEQ ID NO: 39). TheCDR3 regions from the kappa chain clones included QGTYDSSGWYWA (SEQ IDNO: 40) and LGSYSGYIYI (SEQ ID NO: 41).

Plasmids comprising cDNAs encoding rabbit antibodies targeting epitopesof ASPH described in Examples 5-8 are used as a source of nucleic acidscomprising variable regions to generate humanized monoclonal antibodiesthat target at least one epitope in the catalytic domain of ASPH. One ormore codons within the rabbit cDNAs may be altered to represent codonsthat are optimally used in the host cell expression system, to enhanceexpression of the encoded chimeric polypeptide under the control ofoperably-linked promoters and other genetic elements. Random andtargeted mutagenesis of specific residues within the variable regionsmay result in antibodies that have increased affinity to its intendedtarget, and/or reduced affinity to other targets.

Example 14—Generation of Bispecific Antibodies Targeting at Least OneEpitope in the Catalytic Domain of ASPH

Bispecific antibodies combine the structural domains of two distinctmolecules into one molecule with the goal of preserving and perhapsenhancing functional properties of the chimeric molecule compared to itsparent mono-specific molecules (Dahlen E. et al, Bispecific antibodiesin cancer immunotherapy. Therapeutic Advances in Vaccines andImmunotherapy, 2018, 6:(1)3-17). In some cases, bispecific antibodieshave superior therapeutic properties compared to compositions comprisingmixtures of monospecific compounds.

Several classes of immunotherapeutic bispecific antibodies have beenrecognized, including T-cell redirectors, which act on malignant cellsby targeting a tumor antigen and CD3; NK-cell redirectors, which act onmalignant cells targeting a tumor antigen and CD16A; Tumor-targetedimmunomodulators, which direct co-stimulation of tumor-infiltratingimmune cells by targeting a tumor antigen and co-stimulatory molecules,such as CD40 or 4-1BB; and Dual immunomodulators, which simultaneouslyact on two immunomodulatory targets, resulting in blockade of inhibitorytargets, depletion of suppressive cells, or activation of effector cells(See Table 1 of Dahlen et al).

A non-limiting list of exemplary tumor antigens includes CD19, EpCAM,CD20, CD23, BCMA, B7H3, and PSMA.

A non-limiting list of T-cell specific epitopes includes CD3, CD3e,OX40, CD27, ICOS and GITR.

A non-limiting list of co-stimulatory molecules includes CD40 and 4-1BB.

A non-limiting list of immunomodulating targets includes PD-L1, CTLA-4,TGF-0, LAG-2, TIM-3, and OX40.

Bispecific antibodies comprising at least onecomplementarity-determining region (CDR) targeting one or more epitopesof ASPH selected from the group consisting of CDR1, CDR2, and CDR3 fromthe heavy chain or the light chain clones of Example 13 are prepared byfusing rabbit, other non-human, human, or humanized antibodiescomprising these regions with an antibody targeting one or more tumorantigens, T-cell specific epitopes, co-stimulatory molecules, orimmunomodulating targets, as noted above.

Exemplary bi-specific antibodies include a molecule comprising the CDRsof the 5H4/5K3 antibody disclosed herein, where the 5H4 CDR1=NAMC (SEQID NO: 31), CDR2=CIDN (SEQ ID NO: 34), and CDR3=NFNI (SEQ ID NO: 36),and where the 5K3 CDR1=SVYDNNR (SEQ ID NO: 38)), CDR2=LAS (SEQ ID NO:39), CDR3=LGSYSGYIYI (SEQ ID NO: 41) or 9H2/9K1 antibody, where the 9H2CDR1=NAMC (SEQ ID NO: 32), CDR2=CIDN (SEQ ID NO: 34), and CDR3=NFNI (SEQID NO: 36), and the 9K1 CDR1=SVYDNNR (SEQ ID NO: 38), CDR2=LAS (SEQ IDNO: 39), and CDR3=LGSYSGYIYI (SEQ ID NO: 41), combined with an antibodymolecule comprising one or more tumor antigens, T-cell specificepitopes, co-stimulatory molecules, or immunomodulating targets, asnoted above.

An exemplary bispecific antibody of the T-cell redirector class includesan antibody targeting one or more ASPH CDRs with an antibody targetingCD3.

An exemplary bispecific antibody of the NK-cell redirector classincludes an antibody targeting one or more ASPH CDRs with an antibodytargeting CD16A.

An exemplary bispecific antibody of the tumor targeting immunomodularclass includes an antibody targeting one or more ASPH CDRs with anantibody targeting CD40 or 4-1BB.

An exemplary bispecific antibody of the dual immunomodular classincludes an antibody targeting one or more ASPH CDRs with an antibodytargeting PD-L1, PD-1, CTLA-4, TGF-β, LAG-3, TIM-3, or OX40.

Statement Regarding Preferred Aspects are Meant to be Illustrative andnot Limiting as to the Scope of the Invention

While the preferred aspects of the invention have been illustrated anddescribed in detail, it will be appreciated by those skilled in the artthat that various changes can be made therein without departing from thespirit and scope of the invention. Accordingly, the particulararrangements disclosed are meant to be illustrative only and notlimiting as to the scope of the invention, which is to be given the fullbreadth of the appended claims and any equivalent thereof.

BIBLIOGRAPHY Statement Regarding Incorporation by Reference of JournalArticles and Patent Documents

All references, patents, or applications cited herein are incorporatedby reference in their entirety, as if written herein.

Journal Articles

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Wands (2006). “Aspartyl-asparagyl    beta hydroxylase over-expression in human hepatoma is linked to    activation of insulin-like growth factor and notch signaling    mechanisms.” Hepatology 44(2): 446-457.-   Dinchuk, J. E., R. J. Focht, J. A. Kelley, N. L. Henderson, N. I.    Zolotarjova, R. Wynn, N. T. Neff, J. Link, R. M. Huber, T. C.    Burn, M. J. Rupar, M. R. Cunningham, B. H. Selling, J. Ma, A. A.    Stern, G. F. Hollis, R. B. Stein and P. A. Friedman (2002). “Absence    of post-translational aspartyl beta-hydroxylation of epidermal    growth factor domains in mice leads to developmental defects and an    increased incidence of intestinal neoplasia.” J Biol Chem 277(15):    12970-12977.-   Drakenberg, T., P. Fernlund, P. Roepstorff and J. Stenflo (1983).    “beta-Hydroxyaspartic acid in vitamin K-dependent protein C.” Proc    Natl Acad Sci USA 80(7): 1802-1806.-   El Asmar, Z., J. Terrand, M. Jenty, L. Host, M. Mlih, A. Zerr, H.    Justiniano, R. L. Matz, C. Boudier, E. Scholler, J. M. Gamier, D.    Bertaccini, D. Thierse, C. Schaeffer, A. Van Dorsselaer, J. Herz, V.    Bruban and P. Boucher (2016). “Convergent Signaling Pathways    Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and    Extracellular Domains Limit Cellular Cholesterol Accumulation.” J    Biol Chem 291(10): 5116-5127.-   Furler, R. L., D. F. Nixon, C. A. Brantner, A. Popratiloff and C. H.    Uittenbogaart (2018). “TGF-beta Sustains Tumor Progression through    Biochemical and Mechanical Signal Transduction.” Cancers (Basel)    10(6). Gundogan, F., G. Elwood, D. Greco, L. P. Rubin, H.    Pinar, R. I. Carlson, J. R. Wands and S. M. de la Monte (2007).    “Role of aspartyl-(asparaginyl) beta-hydroxylase in placental    implantation: Relevance to early pregnancy loss.” Hum Pathol 38(1):    50-59.-   Iwagami, Y., S. Casulli, K. Nagaoka, M. Kim, R. I. Carlson, K.    Ogawa, M. S. Lebowitz, S. Fuller, B. Biswas, S. Stewart, X. Dong, H.    Ghanbari and J. R. Wands (2017). “Lambda phage-based vaccine induces    antitumor immunity in hepatocellular carcinoma.” Heliyon 3(9):    e00407.-   Lavaissiere, L., S. Jia, M. Nishiyama, S. de la Monte, A. M.    Stern, J. R. Wands and P. A. Friedman (1996). “Overexpression of    human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular    carcinoma and cholangiocarcinoma.” J Clin Invest 98(6): 1313-1323.-   Noda, T., M. Shimoda, V. Ortiz, A. E. Sirica and J. R. Wands (2012).    “Immunization with aspartate-beta-hydroxylase-loaded dendritic cells    produces antitumor effects in a rat model of intrahepatic    cholangiocarcinoma.” Hepatology 55(1): 86-97.-   Revskaya, E., Z. Jiang, A. Morgenstern, F. Bruchertseifer, M.    Sesay, S. Walker, S. Fuller, M. S. Lebowitz, C. Gravekamp, H. A.    Ghanbari and E. Dadachova (2017). “A Radiolabeled Fully Human    Antibody to Human Aspartyl (Asparaginyl) beta-Hydroxylase Is a    Promising Agent for Imaging and Therapy of Metastatic Breast    Cancer.” Cancer Biother Radiopharm 32(2): 57-65.-   Tong, M., J. S. Gao, D. Borgas and S. M. de la Monte (2013).    “Phosphorylation Modulates Aspartyl-(Asparaginyl)-beta Hydroxylase    Protein Expression, Catalytic Activity and Migration in Human    Immature Neuronal Cerebellar Cells.” Cell Biol (Henderson, Nev.)    6(2).-   Wu, G., Z. Ma, Y. Cheng, W. Hu, C. Deng, S. Jiang, T. Li, F. Chen    and Y. Yang (2018). “Targeting Gas6/TAM in cancer cells and tumor    microenvironment.” Mol Cancer 17(1): 20.-   Yang, H., K. Song, T. Xue, X. P. Xue, T. Huyan, W. Wang and H. Wang    (2010). “The distribution and expression profiles of human    Aspartyl/Asparaginyl beta-hydroxylase in tumor cell lines and human    tissues.” Oncol Rep 24(5): 1257-1264.-   Yeung, Y. A., A. H. Finney, I. A. Koyrakh, M. S. Lebowitz, H. A.    Ghanbari, J. R. Wands and K. D. Wittrup (2007). “Isolation and    characterization of human antibodies targeting human aspartyl    (asparaginyl) beta-hydroxylase.” Hum Antibodies 16(3-4): 163-176.-   PDB ID SJZZ: McDonough, M. A., Pfeffer, I., Munzel, M. (2016)    Aspartyl/Asparaginyl beta-hydroxylase (AspH)oxygenase and TPR    domains in complex with manganese, N-oxalylglycine and cyclic    peptide substrate mimic of factor X. DOI: 10.2210/pdb5JZZ/pdb.    Deposited as PDB ID SJZZ on 2016-05-16, Released on 2017 Jun. 6;    Replaced by PDB ID 6RK9 on 2019-05-07.-   Dahlen E., Veltonmaki, and Norten, P. (2018) Bispecific antibodies    in cancer immunotherapy. Therapeutic Advances in Vaccines and    Immunotherapy 6(1): 3-17.

What is claimed is:
 1. A composition comprising two or more humanizedantibodies or fragments or variants thereof, wherein one humanizedantibody or fragment or variant thereof, targets at least one epitope ofASPH and another humanized antibody or fragment or variant thereoftargets at least one epitope selected from the group consisting of theT-cell redirector class, comprising an antibody targeting one or moreASPH CDRs and an antibody targeting CD3; the NK-cell redirector class,comprising an antibody targeting one or more ASPH CDRs and an antibodytargeting CD16A; the tumor targeting immunomodular class, comprising anantibody targeting one or more ASPH CDRs and an antibody targeting CD40or 4-1BB; and the dual immunomodular class, comprising an antibodytargeting one or more ASPH CDRs and an antibody targeting PD-L1, PD-1,CTLA-4, TGF-β, LAG-3, TIM-3, or OX40.
 2. The composition of claim 1,wherein said humanized antibody or fragment or variant thereof, whichbinds to one or more peptide epitopes of human aspartyl (asparaginyl)β-hydroxylase (ASPH) comprises: a recombinant heavy chain and arecombinant light chain, each heavy and each light chain comprising 3complementarity-determining regions (CDRs), or fragment or variantthereof, which binds to one or more peptide epitopes of human aspartyl(asparaginyl) β-hydroxylase (ASPH), wherein at least one of said peptideepitopes comprises at least 4 consecutive amino acid residues locatedwithin or adjacent to a position in the catalytic domain of ASPH that iswithin 30 amino acids of the C-terminus of human ASPH, corresponding tothe sequence QDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions729-758 of SEQ ID NO: 1;

wherein said antibody or fragment or variant thereof contains one ormore conservative amino acid substitutions in which the functionalactivity relating to binding of the antibody or fragment thereof to anepitope of ASPH is retained; wherein said antibody comprises arecombinant heavy chain comprising a CDR1 comprising a sequence selectedfrom the group consisting of NFMC represented by SEQ ID NO: 31, andNAMC represented by SEQ ID NO: 32;

a CDR2 comprising a sequence selected from the group consisting of CIYFrepresented by SEQ ID NO: 33, and CIDN represented by SEQ ID NO: 34;

 and a CDR3 comprising a sequence selected from the group consisting ofDGPGSISWKI represented by SEQ ID NO: 35, and NFNIrepresented by SEQ ID NO: 36;

wherein said antibody comprises a recombinant light chain comprising aCDR1 comprising a sequence selected from the group consisting of SVYSKNRrepresented by SEQ ID NO: 37, and SVYDNNR represented by SEQ ID NO: 38;

a CDR2 comprising the sequence LAS represented by SEQ ID NO: 39;

 and a CDR3 comprising a sequence selected from the group consisting ofQGTYDSSGWYWA represented by SEQ ID NO: 40, and LGSYSGYIYIrepresented by SEQ ID NO:
 41.


3. The composition of claim 2, wherein said humanized antibody orfragment or variant that targets at least one epitope of ASPH binds toone or more peptides selected from the group consisting of (a) a peptidecomprising 29 amino acids with Cysteine at its amino terminus, plus 28amino acids corresponding to positions 731-758 at the C-terminal end ofhuman ASPH represented by SEQ ID NO: 1, with the Threonine at relativeposition 19, corresponding to position 748 of human ASPH,phosphorylated, as CASSFRLIFIVDVWHPEL-T(PO₃H₂)-PQQRRSLPAIrepresented by SEQ ID NO: 19;

and (b) a peptide comprising 29 amino acids with Cysteine at its aminoterminus, plus 28 amino acids corresponding to positions 731-758 at theC-terminal end of human ASPH represented by SEQ ID NO: 1, asCASSFRLIFIVDVWHPELTPQQRRSLPAI, represented by SEQ ID NO:
 20.


4. The composition of claim 2, wherein said antibody humanized antibodyor fragment or variant that targets at least one epitope of ASPHcomprises a recombinant heavy chain and a recombinant light chain, or afragment thereof, which binds to one or more peptide epitopes of humanaspartyl (asparaginyl) β-hydroxylase (ASPH), wherein at least one ofsaid peptide epitopes is located within or adjacent to a position in thecatalytic domain of ASPH that is within 30 amino acids of the C-terminusof human ASPH, corresponding to the sequenceQDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions729-758 of SEQ ID NO: 1;

wherein said antibody binds to an epitope comprising at least 4consecutive amino acid residues located within 30 amino acids from theC-terminal end of human ASPH represented by SEQ ID NO: 1; wherein saidepitope comprising at least 4 consecutive amino acid residues locatedwithin 30 amino acids from the C-terminal end of human ASPH comprisesthe consecutive amino acid residues selected from the group consistingof PELT represented by SEQ ID NO: 42, ELTP represented by SEQ ID NO: 43,LTPQ represented by SEQ ID NO: 44, TPQQ represented by SEQ ID NO: 45,PQQR represented by SEQ ID NO: 46, QQRR represented by SEQ ID NO: 47,QRRS represented by SEQ ID NO: 48, RRSL represented by SEQ ID NO: 49,RSLP represented by SEQ ID NO: 50, SLPA represented by SEQ ID NO: 51,and LPAI represented by SEQ ID NO:
 52.


5. A composition comprising at least one humanized bispecific antibody,or fragment or variant thereof, wherein said antibody or fragment orvariant thereof comprises one or more complementarity determiningregions (CDRs) derived from a non-human source targeting one or morepeptide epitopes located within or adjacent to the catalytic domain ofASPH, and an antibody targeting other epitopes selected from the groupconsisting of the T-cell redirector class, comprising an antibodytargeting one or more ASPH CDRs and an antibody targeting CD3; theNK-cell redirector class, comprising an antibody targeting one or moreASPH CDRs and an antibody targeting CD16A; the tumor targetingimmunomodular class, comprising an antibody targeting one or more ASPHCDRs and an antibody targeting CD40 or 4-1BB; and the dual immunomodularclass, comprising an antibody targeting one or more ASPH CDRs and anantibody targeting PD-L1, PD-1, CTLA-4, TGF-β, LAG-3, TIM-3, or OX40. 6.The composition of claim 5, wherein said humanized antibody or fragmentor variant thereof, which binds to one or more peptide epitopes of humanaspartyl (asparaginyl) β-hydroxylase (ASPH) comprises: a recombinantheavy chain and a recombinant light chain, each heavy and each lightchain comprising 3 complementarity-determining regions (CDRs), orfragment or variant thereof, which binds to one or more peptide epitopesof human aspartyl (asparaginyl) β-hydroxylase (ASPH), wherein at leastone of said peptide epitopes comprises at least 4 consecutive amino acidresidues located within or adjacent to a position in the catalyticdomain of ASPH that is within 30 amino acids of the C-terminus of humanASPH, corresponding to the sequence QDASSFRLIFIVDVWHPELTPQQRRSLPAIrepresented by positions 729-758 of SEQ ID NO: 1;

wherein said antibody or fragment or variant thereof contains one ormore conservative amino acid substitutions in which the functionalactivity relating to binding of the antibody or fragment thereof to anepitope of ASPH is retained; wherein said antibody comprises arecombinant heavy chain comprising a CDR1 comprising a sequence selectedfrom the group consisting of NFMC represented by SEQ ID NO: 31, and NAMCrepresented by SEQ ID NO: 32;

a CDR2 comprising a sequence selected from the group consisting of CIYFrepresented by SEQ ID NO: 33, and CIDN represented by SEQ ID NO: 34;

 and a CDR3 comprising a sequence selected from the group consisting ofDGPGSISWKI represented by SEQ ID NO: 35, and NFNIrepresented by SEQ ID NO: 36;

wherein said antibody comprises a recombinant light chain comprising aCDR1 comprising a sequence selected from the group consisting of SVYSKNRrepresented by SEQ ID NO: 37, and SVYDNNR represented by SEQ ID NO: 38;

a CDR2 comprising the sequence LAS represented by SEQ ID NO: 39;

 and a CDR3 comprising a sequence selected from the group consisting ofQGTYDSSGWYWA represented by SEQ ID NO: 40, and LGSYSGYIYIrepresented by SEQ ID NO:
 41.


7. The composition of claim 6, wherein said humanized antibody orfragment or variant that targets at least one epitope of ASPH binds toone or more peptides selected from the group consisting of (a) a peptidecomprising 29 amino acids with Cysteine at its amino terminus, plus 28amino acids corresponding to positions 731-758 at the C-terminal end ofhuman ASPH represented by SEQ ID NO: 1, with the Threonine at relativeposition 19, corresponding to position 748 of human ASPH,phosphorylated, as CASSFRLIFIVDVWHPEL-T(PO₃H₂)-PQQRRSLPAIrepresented by SEQ ID NO: 19;

and (b) a peptide comprising 29 amino acids with Cysteine at its aminoterminus, plus 28 amino acids corresponding to positions 731-758 at theC-terminal end of human ASPH represented by SEQ ID NO: 1, asCASSFRLIFIVDVWHPELTPQQRRSLPAI, represented by SEQ ID NO:
 20.


8. The composition of claim 6, wherein said antibody humanized antibodyor fragment or variant that targets at least one epitope of ASPHcomprises a recombinant heavy chain and a recombinant light chain, or afragment thereof, which binds to one or more peptide epitopes of humanaspartyl (asparaginyl) hydroxylase (ASPH), wherein at least one of saidpeptide epitopes is located within or adjacent to a position in thecatalytic domain of ASPH that is within 30 amino acids of the C-terminusof human ASPH, corresponding to the sequenceQDASSFRLIFIVDVWHPELTPQQRRSLPAI represented by positions729-758 of SEQ ID NO: 1;

wherein said antibody binds to an epitope comprising at least 4consecutive amino acid residues located within 30 amino acids from theC-terminal end of human ASPH represented by SEQ ID NO: 1; wherein saidepitope comprising at least 4 consecutive amino acid residues locatedwithin 30 amino acids from the C-terminal end of human ASPH comprisesthe consecutive amino acid residues selected from the group consistingof PELT represented by SEQ ID NO: 42, ELTP represented by SEQ ID NO: 43,LTPQ represented by SEQ ID NO: 44, TPQQ represented by SEQ ID NO: 45,PQQR represented by SEQ ID NO: 46, QQRR represented by SEQ ID NO: 47,QRRS represented by SEQ ID NO: 48, RRSL represented by SEQ ID NO: 49,RSLP represented by SEQ ID NO: 50, SLPA represented by SEQ ID NO: 51,and LPAI represented by SEQ ID NO: 52.